Methods of treating rheumatoid arthritis with 7C10 and 16C10 CD80-specific antibodies

ABSTRACT

The present invention relates to the identification of antibodies which are specific to human B7.1 antigen (CD80) and which are capable of inhibiting the binding of B7.1 to a CD28 receptor and which are not capable of inhibiting the binding of B7.1 to a CTLA-4 receptor. Two of these antibodies, 16C10 and 7C10, significantly inhibit the production of IL-2, in spite of the existence of a second activating ligand B7.2 (CD86). Blocking of the primary activation signal between CD28 and B7.1 (CD80) with these antibodies while allowing the unimpaired or coincident interaction of CTLA-4 and B7.1 and/or B7.2 represents a combined antagonistic effect on positive co-stimulation with an agonistic effect on negative signalling. These antibodies may be used as specific immunosuppressants, e.g., for the treatment of autoimmune diseases and to prevent organ transplant rejection.

RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 11/464,123, filedAug. 11, 2006, which issued as U.S. Pat. No. 7,414,123, which is acontinuation of U.S. application Ser. No. 09/576,424, filed May 22,2000, and issued as U.S. Pat. No. 7,175,847, which iscontinuation-in-part of U.S. application Ser. No. 08/746,361, filed Nov.8, 1996, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 08/487,550, filed Jun. 7, 1995, and issued as U.S.Pat. No. 6,113,898. Each of the above-identified applications isincorporated by reference in its entirety herein.

FIELD OF THE INVENTION

The present invention relates to the identification and use ofmonoclonal antibodies which are specific to B7.1 antigens (CD80). Morespecifically, the present invention relates to the identification anduse of monoclonal antibodies or PRIMATIZED® forms thereof which arecapable of inhibiting the binding of human B7.1 antigen to a CD28receptor and which are not capable of inhibiting the binding of B7.1 toa CTLA-4 receptor. Thus, the invention relates to the identification anduse of monoclonal antibodies and PRIMATIZED® forms thereof whichrecognize specific sites on the B7.1 antigen which are exclusive ofCTLA-4 receptor binding.

The invention further relates to monoclonal antibodies or PRIMATIZED®forms thereof which recognize specific sites on the human B7.1 antigenand are capable of inhibiting IL-2 production.

Also, the present invention relates to pharmaceutical compositionscontaining monoclonal or PRIMATIZED® antibodies specific to human B7.1and their use as immunosuppressants by modulating the B7:CD28 pathway,e.g., for the treatment of autoimmune disorders, and the prevention oforgan rejection.

BACKGROUND OF THE INVENTION

The clinical interface between immunology, hematology, and oncology haslong been appreciated. Many conditions treated by the hematologist oroncologist have either an autoimmune or immuno-deficient component totheir pathophysiology that has led to the widespread adoption ofimmunosuppressive medications by hematologists, whereas oncologists havesought immunologic adjuvants that might enhance endogenous immunity totumors. To date, these interventions have generally consisted ofnonspecific modes of immunosuppression and immune stimulation. Inaddition to the limited efficacy of these interventions, toxicitiessecondary to their nonspecificity have also limited their overallsuccess. Therefore, alternative strategies have been sought.

Elucidation of the functional role of a rapidly increasing number ofcell surface molecules has contributed greatly to the integration ofimmunology with clinical hematology and oncology. Nearly 200 cellsurface antigens have been identified on cells of the immune andhematopoietic systems (Schlossman S F, Boumsell L, Gilks J M, Harlan T,Kishimoto, C Morimoto C, Ritz J., Shaw S, Silverstein R L, Springer T A,Tedder T F, Todd R F:CD antigens (1993), Blood 83:879, 1994). Theseantigens represent both lineage-restricted and more widely distributedmolecules involved in a variety of processes, including cellularrecognition, adhesion, induction and maintenance of proliferation,cytokine secretion, effector function, and even cell death. Recognitionof the functional attributes of these molecules has fostered novelattempts to manipulate the immune response. Although molecules involvedin cellular adhesion and antigen-specific recognition have previouslybeen evaluated as targets of therapeutic immunologic intervention,recent attention has focused on a subgroup of cell surface moleculestermed co-stimulatory molecules (Bretscher P: “The two-signal model oflymphocyte activation twenty-one years later.” Immunol. Today 13:73(1992); Jenkins M K, Johnson J G: “Molecules involved in T-cellco-stimulation.” Curr Opin Immunol 5:351 (1993); Geppert T, Davis L. GurH. Wacholtz M. Lipsky P: “Accessory cell signals involved in T-cellactivation.” Immunol Rev 117:5 (1990); Weaver C T, Unanue E R: “Theco-stimulatory function of antigen-presenting cells.” Immunol Today11:49 (1990); Stennam R M, Young J W: “Signals arising fromantigen-presenting cells.” Curr Opin Immunol 3:361 (1991)).Co-stimulatory molecules do not initiate but rather enable thegeneration and amplification of antigen-specific T-cell responses andeffector function (Bretscher P: “The two-signal model of lymphocyteactivation twenty-one years later.” Immunol. Today 13:73 (1992); JenkinsM K, Johnson J G: “Molecules involved in T-cell co-stimulation.” CurrOpin Immunol 5:351 (1993); Geppert T, Davis L. Gur H. Wacholtz M. LipskyP: “Accessory cell signals involved in T-cell activation.” Immunol Rev117:5 (1990); Weaver C T, Unanue E R: “The co-stimulatory function ofantigen-presenting cells.” Immunol Today 11:49, (1990); Stennam R M,Young J W: “Signals arising from antigen-presenting cells.” Curr OpinImmunol 3:361 (1991); June C H, Bluestone J A, Linsley P S, Thompson CD: “Role of the CD28 receptor in T-cell activation.” Immunol Today15:321 (1994)).

Recently, one specific co-stimulatory pathway termed B7:CD28 has beenstudied by different research groups because of its significant role inB- and T-cell activation (June C H, Bluestone J A, Linsley P S, ThompsonC D: “Role of the CD28 receptor in T-cell activation.” Immunol Today15:321 (1994); June C H, Ledbetter J A: “The role of the CD28 receptorduring T-cell responses to antigen.” Annu Rev Immunol 11:191 (1993);Schwartz R H: “Co-stimulation of T lymphocytes: The role of CD28,CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy.” Cell71:1065-1068 (1992); Jenkins M K, Taylor P S, Norton S D, Urdahl K B:“CD28 delivers a co-stimulatory signal involved in antigen-specific IL-2production by human T cells.” Journal of Immunology 147:2461-2466(1991)). Since this ligand:receptor pathway was discovered four yearsago, a large body of evidence has accumulated suggesting that B7:CD28interactions represent one of the critical junctures in determiningimmune reactivity versus anergy (June C H, Bluestone J A, Linsley P S,Thompson C D: “Role of the CD28 receptor in T-cell activation.” ImmunolToday 15:321 (1994); June C H, Ledbetter J A: “The role of the CD28receptor during T-cell responses to antigen.” Annu Rev Immunol 11:191(1993); Schwartz R H: “Co-stimulation of T lymphocytes: The role ofCD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy.”Cell 71:1065-1068 (1992); Cohen J: “Mounting a targeted strike onunwanted immune responses” (news; comment). Science 257:751 (1992);Cohen J: “New protein steals the show as ‘co-stimulator’ of T cells”(news; comment). Science 262:844 (1993)).

In particular, the role of the human B7 antigens, i.e., human B7.1(CD80) and B7.2 (CD86), has been reported to play a co-stimulatory rolein T-cell activation. See, e.g., Gimmi C D, Freeman, G J, Gribben J G,Sugita K, Freedman A S, Morimoto C, Nadler L M: “B-cell surface antigenB7 provides a costimulatory signal that induces T cells to proliferateand secrete interleukin 2.” Proc. Natl. Acad. Sci. (USA) 88:6575-6579(1991).

1. B7.1 and B7.2 Co-Stimulatory Role in T Cell Activation

The elaboration of a successful immune response depends on a series ofspecific interactions between a T cell and an antigen presenting cell.Although the essential first step in this process depends upon thebinding of antigen to the T cell receptor, in the context of the MHCclass II molecule (Lane, P. J. L., F. M. McConnell, G. L. Schieven, E.A. Clark, and J. A. Ledbetter, (1990), “The Role of Class II Moleculesin Human B Cell Activation.” The Journal of Immunology 144:3684-3692),this interaction alone is not sufficient to induce all the eventsnecessary for a sustained response to a given antigen (Schwartz, R. H.(1990), “A Cell Culture Model for T Lymphocyte Clonal Anergy.” Science248:1349; Jenkins, M. K. (1992), “The Role of Cell Division in theInduction of Clonal Anergy.” Immunology Today 13:69; Azuma, M., M.Cayabyab, D. Buck, J. H. Phillips, and L. L. Lanier (1992), “Involvementof CD28 in MHC-unrestricted Cytotoxicity Mediated by a Human NaturalKiller Leukemia Cell Line.” The Journal of Immunology 149:1115-1123;Azuma, M., M. Cayabyab, D. Buck, J. H. Phillips, and L. L. Lanier(1992), “CD28 Interaction with B7 Costimulates Primary AllogeneicProliferative Responses and Cytotoxicity Mediated by Small Resting TLymphocytes.” J. Exp. Med. 175:353-360); S. D. Norton, L. Zuckerman, K.B. Urdahl, R. Shefner, J. Miller, and M. K. Jenkins (1992), “The CD28Ligand, B7, Enhances IL-2 Production by Providing a Costimulatory Signalto T Cells.” The Journal of Immunology 149:1556-1561; R. H. Schwartz(1992), “Costimulation of T Lymphocytes: The Role of CD28, CTLA-4, andB7/BB1 in Interleukin-2 Production and Immunotherapy.” Cell71:1065-1068).

The involvement of certain other co-stimulatory molecules is necessary(Norton, S. D., L. Zuckerman, K. B. Urdahl, R. Shefner, J. Miller, andM. K. Jenkins (1992), “The CD28 Ligand, B7, Enhances IL-2 Production byProviding A Costimulatory Signal to T Cells.” The Journal of Immunology149:1556-1561)), “The homodimers CD28 and CTLA-4 expressed on T cells”(June, C. H., J. A. Ledbetter, P. S. Linsley, and C. B. Thompson (1990),“Role of the CD28 Receptor in T-Cell Activation.” Immunology Today11:211-216; Linsley, P. S., W. Brady, M. Umes, L. S. Grosmaire, N. K.Damle, and J. A. Ledbetter (1991), “CTLA-4 is a Second Receptor for theB Cell Activation Antigen B7.” J. Exp. Med. 174:561)), together withB7.1 (CD80) and B7.2 (CD86) expressed on antigen presenting cells, aremajor pairs of co-stimulatory molecules necessary for a sustained immuneresponse (Azuma, M., H. Yssel, J. H. Phillips, H. Spits, and L. L.Lanier (1993), “Functional Expression of B7/BB1 on Activated TLymphocytes.” J. Exp. Med. 177:845-850; Freeman, G. J., A. S. Freedman,J. M. Segil, G. Lee, J. F. Whitman, and L M. Nadler (1989), “B7, A NewMember of the Ig Superfamily with Unique Expression on Activated andNeoplastic B Cells.” The Journal of Immunology 143:2714-2722; Hathcock,K. S., G. Laslo, H. B. Dickler, J. Bradshaw, P. Linsley, and R. J. Hodes(1993), “Identification of an Alternative CTLA-4 Ligand Costimulatoryfor T Cell Activation.” Science 262:905-911; Hart, D. N. J., G. C.Starling, V. L. Calder, and N. S. Fernando (1993), “B7/BB-1 is aLeucocyte Differentiation Antigen on Human Dendritic Cells Induced byActivation.” Immunology 79:616-620). It can be shown in vitro that theabsence of these co-stimulatory signals leads to an aborted T cellactivation pathway and the development of unresponsiveness to thespecific antigen, or anergy. (See, e.g., Harding, F. A., J. G. McArthur,J. A. Gross, D. M. Raulet, and J. P. Allison (1992), “CD28 MediatedSignaling Co-stimulates Murine T Cells and Prevents Induction of Anergyin T Cell Clones.” Nature 356:607-609; Gimmi, C. D., G. J. Freeman, J.G. Gribben, G. Gray, and L. M. Nadler (1993); “Human T-Cell ClonalAnergy is Induced by Antigen Presentation in the Absence of B7Costimulation.”, Proc. Natl. Acad. Sci. 90:6586-6590; Tan, P., C.Anasefti, J. A. Hansen, J. Melrose, M. Brunvand, J. Bradshaw, J. A.Ledbetter, and P. S. Linsley (1993), “Induction of Alloantigen-specificHyporesponsiveness in Human T Lymphocytes by Blocking Interaction ofCD28 with Its Natural Ligand B7/BB1.” J. Exp. Med. 177:165-173).Achievement of in vivo tolerance constitutes a mechanism forimmunosuppression and a viable therapy for organ transplant rejectionand for the treatment of autoimmune diseases. This has been achieved inexperimental models following the administration of CTLA-4Ig (Lenschow,D. J., Y. Zeng, R. J. Thistlethwaite, A. Montag, W. Brady, M. G. Gibson,P. S. Linsley, and J. A. Bluestone (1992), “Long-Term Survival ofXenogeneic Pancreatic Islet Grafts Induced by CTLA-4Ig.” Science257:789-795).

The molecules B7.1 and B7.2 can bind to either CD28 or CTLA-4, althoughB7.1 binds to CD28 with a Kd of 200 Nm and to CTLA-4 with a 20-foldhigher affinity (Linsley, P. S., E. A. Clark, and J. A. Ledbetter(1990), “T-Cell Antigen CD28 Mediates Adhesion with B Cells byInteracting with Activation Antigen B7/BB-1.” Proc. Natl. Acad. Sci.87:5031-5035; Linsley et al (1993), “The Role of the CD28 receptorduring T cell responses to antigen,” Annu. Rev. Immunol. 11:191-192;Linesley et al (1993), “CD28 Engagement by B7/BB-1 Induces TransientDown-Regulation of CD28 Synthesis and Prolonged Unresponsiveness to CD28Signaling,” The Journal of Immunology 150:3151-3169). B7.1 is expressedon activated B cells and interferon induced monocytes, but not resting Bcells (Freeman, G. J., G. S. Gray, C. D. Gimmi, D. B. Lomarrd, L-J.Zhou, M. White, J. D. Fingeroth, J. G. Gribben, and L M. Nadler (1991).“Structure, Expression and T Cell Costimulatory Activity of the MurineHomologue of the Human B Lymphocyte Activation Antigen B7,” J. Exp.Med., 174:625-631). B7.2, on the other hand, is constitutively expressedat very low levels on resting monocytes, dendritic cells and B cells,and its expression is enhanced on activated T cells, NK cells and Blymphocytes (Azuma, M. D. Ito, H. Yagita, K. Okumura, J. H. Phillips, L.L. Lanier, and C. Somoza 1993, “B70 Antigen is a Second Ligand forCTLA-4 and CD28,” Nature, 366:76-79). Although B7.1 and B7.2 can beexpressed on the same cell type, their expression on B cells occurs withdifferent kinetics (Lenschow, D. J., G. H. Su, L. A. Zuckerman, N.Nabavi, C. L. Jellis, G. S. Gray, J. Miller, and J. A. Bluestone (1993),“Expression and Functional Significance of an Additional Ligand forCTLA-4,” Proc. Natl. Acad. Sci., USA, 90:11054-11058; Boussiotis, V. A.,G. J. Freeman, J. G. Gribben, J. Daley, G. Gray, and L. M. Nadler(1993), “Activated Human B Lymphocytes Express Three CTLA-4Counter-receptors that Co-stimulate T-Cell Activation.” Proc. Natl.Acad. Sci., USA, 90:11059-11063). Further analysis at the RNA level hasdemonstrated that B7.2 mRNA is constitutively expressed, whereas B7.1mRNA is detected 4 hours after activation and initial low levels of B7.1protein are not detectable until 24 hours after stimulation (Boussiotis,V. A., G. J. Freeman, J. G. Gribben, J. Daley, G. Gray, and L. M. Nadler(1993), “Activated Human B Lymphocytes Express Three CTLA-4Counter-receptors that Co-stimulate T-Cell Activation,” Proc. Natl.Acad. Sci., USA, 90:11059-11063). CTLA-4/CD28 counter receptors,therefore, may be expressed at various times after B Cell activation.

More recently, it has been suggested that the second T cell associatedco-receptor CTLA-4 apparently functions as a negative modulator tooverride and prevent a runaway immune system (Krummel M, Allison J:“CD28 and CTLA-4 have opposing effects on the response of T cells tostimulation.” J. Exp. Med. 182:459-466 (1995)). The CTLA-4 receptorplays a critical role in down regulating the immune response, asevidenced in CTLA-4 knockout mice. Knockout mice born without theability to express the CTLA-4 gene die within 3-4 weeks of severelymphoproliferative disorder (Tivol E A, Borriello G, Schweitzer A N,Lynch W P, Bluestone J A, Sharpe A H: “Loss of CTLA-4 leads to massivelymphoproliferation and fatal multiorgan tissue destruction, revealing acritical negative regulatory role of CTLA-4.” Immunity 3:541-547(1995)). CTLA-4 is thought to function through signaling mechanismslinked to induction of apoptosis (Gribben J G, Freeman G J, Boussiotis VA, Rennert P, Jellis C L, Greenfield E, Barber M, Restivo Jr. V A, Ke X,Gray G S, Nadler L M: “CTLA-4 mediates antigen specific apoptosis ofhuman T cells.” Proc. Natl. Acad. Sci. USA 92:811-815 (1995)), triggeredthrough as yet undefined ligand binding to specific cites on thereceptor. It has been shown in vitro that the blocking of the B7.1/B7.2dependent co-stimulatory signals in various ways leads to an aborted Tcell activating pathway and the development of unresponsiveness to thespecific antigen (Lederman S, Chess L, Yellin M J: “Murine monoclonalantibody (5c8) recognizes a human glycoprotein on the surface ofT-lymphocytes, compositions containing same.” U.S. Pat. No. 5,474,771(Dec. 12, 1995); Linsley P S, Ledbetter J A, Damle N K, Brady W:“Chimeric CTLA4 receptor and methods for its use,” U.S. Pat. No.5,434,131 (Jul. 18, 1995); Harding, 1992; Gimmi C D, Freeman G J,Bribben J G, Gray G, Nadler L M: “Human T-cell clonal anergy is inducedby antigen presentation in the absence of B7 costimulation.” Proc. Natl.Acad. Sci. (USA) 90:6586-6590 (1993); Tan P, Anasetti C, Hansen J A,Melrose J, Brunvand M, Bradshaw J, Ledbetter J A, Linsley P S:“Induction of alloantigen-specific hyporesponsiveness in human Tlymphocytes by blocking interaction of CD28 with its natural ligandB7/BB1.” J. Exp. Med. 177:165-173 (1993)). Achievement of in vivotolerance, anergy, or depleting of antigen-specific T cells wouldconstitute a mechanism for immunosuppression and a viable therapy fororgan transplant rejection or plausible treatment for autoimmunediseases.

The differential temporal expression of B7.1 and B7.2 suggests that theinteraction of these two molecules with CTLA-4 and/or CD28 deliverdistinct but related signals to the T cell (LaSalle, J. M., P. J.Tolentino, G. J. Freeman, L. M. Nadler, and D. A. Hafler, (1992), “CD28and T Cell Antigen Receptor Signal Transduction Coordinately RegulateInterleukin 2 Gene Expression In Response to Superantigen Stimulation,”J. Exp. Med., 176:177-186; Vandenberghe, P., G. J. Freeman, L. M.Nadler, M. C. Fletcher, M. Kamoun, L. A. Turka, J. A. Ledbetter, C. B.Thompson, and C. H. June (1992), “Antibody and B7/BB1-mediated Ligationof the CD28 Receptor Induces Tyrosine Phosphorylation in Human T Cells,”The Journal of Experimental Medicine 175:951-960)). The exact signalingfunctions of CTLA-4 and CD28 on the T cell are currently unknown(Janeway, C. A., Jr. and K. Bottomly, (1994), “Signals and Signs forLymphocyte Responses,” Cell 76.275285). However, it is possible that oneset of receptors could provide the initial stimulus for T cellactivation and the second, a sustained signal to allow furtherelaboration of the pathway and clonal expansion to take place (Linsley,P. S., J. L. Greene, P. Tan, J. Bradshaw, J. A. Ledbetter, C. Anasetti,and N. K. Damle, (1992), “Coexpression and Functional Cooperation ofCTLA-4 and CD28 on Activated T Lymphocytes,” J. Exp. Med.176:1595-1604). The current data supports the two-signal hypothesisproposed by Jenkins and Schwartz (Schwartz, R. H. (1990), “A CellCulture Model for T Lymphocyte Clonal Anergy,” Science 248:1349;Jenkins, M. K., (1992), “The Role of Cell Division in the Induction ofClonal Anergy,” Immunology Today 13:69)) that both a TCR andco-stimulatory signal are necessary for T cell expansion, lymphokinesecretion and the full development of effector function (Greenan, V. andG. Kroemer (1993), “Multiple Ways to Cellular Immune Tolerance,”Immunology Today 14:573). The failure to deliver the second signalresults in the inability of T cells to secrete IL-2 and renders the cellunresponsive to antigen.

Structurally, both. B7.1 and B7.2 contain extracellular immunoglobulinsuperfamily V and C-like domains, a hydrophobic transmembrane region anda cytoplasmic tail (Freeman, G. J., J. G. Gribben, V. A. Boussiotis, J.W. Ng, V. Restivo, Jr., L. A. Lombard, G. S. Gray, and L. M. Nadler(1993), “Cloning of B7.2: A CTLA-4 Counter-receptor that Co-stimulatesHuman T Cell Proliferation,” Science 262:909). Both B7.1 and B7.2 areheavily glycosylated. B7.1 is a 44-54 kD glycoprotein comprised of a 223amino acid extracellular domain, a 23 amino acid transmembrane domain,and a 61 amino acid cytoplasmic tail. B7.1 contains 3 potential proteinkinase phosphorylation sites. (Azuma, M., H. Yssel, J. H. Phillips, H.Spits, and L. L. Lanier, (1993), “Functional Expression of B7/BB1 onActivated T Lymphocytes,” J. Exp. Med. 177:845-850). B7.2 is a 306 aminoacid membrane glycoprotein. It consists of a 220 amino acidextracellular region, a 23 amino acid hydrophobic transmembrane domainand a 60 amino acid cytoplasmic tail (Freeman, G. J., A. S. Freedman, J.M. Segil, G. Lee, J. F. Whitman, and L M. Nadler (1989), “B7, A NewMember of the Ig Superfamily with Unique Expression on Activated andNeoplastic B Cells,” The Journal of Immunology 143:2714-2722). Althoughboth B7.1 and B7.2 genes are localized in the same chromosomal region(Freeman, G. J., D. B. Lombard, C. D. Gimmi, S. A. Brod, L Lee, J. C.Laning, D. A. Hafler, M. E. Dorf, G. S. Gray, H. Reiser, C. H. June, C.B. Thompson, and L. M. Nadler (1992), “CTLA-4 and CD28 mRNA areCoexpressed in Most T Cells After Activation,” The Journal of Immunology149:3795-3801; Schwartz, R. H. (1992), “Costimulation of T Lymphocytes:The Role of CD28, CTLA-4, and B7/BB1” in Selvakumar, A., B. K. Mohanraj,R. L. Eddy, T. B. Shows, P. C. White, C. Perrin, and B. Dupont (1992),“Genomic Organization and Chromosomal Location of the Human GeneEncoding the B-Lymphocyte Activation Antigen B7,” Immunogenetics36:175-181), these antigens do not share a high level of homology. Theoverall homology between B7.1 and B7.2 is 26% and between murine B7.1and human B7.1 is 27% (Azuma, M., H. Yssel, J. H. Phillips, H. Spits,and L. L. Lanier (1993), “Functional Expression of B7/BB1 on Activated TLymphocytes,” J. Exp. Med. 177:845-850; Freeman, G. J., A. S. Freedman,J. M. Segil, G. Lee, J. F. Whitman, and LM. Nadler (1989), “B7, A NewMember of the Ig Superfamily with Unique Expression on Activated andNeoplastic B Cells,” The Journal of Immunology 143:2714-2722). Althoughalignment of human B7.1 human B7.2 and murine B7.1 sequences shows fewstretches of lengthy homology, it is known that all three molecules bindto human CTLA-4 and CD28. Thus, there is most likely a common, orclosely homologous region shared by the three molecules that may beeither contiguous or conformational. This region may constitute thebinding site of the B7.1 and B7.2 molecules to their counter-receptors.Antibodies raised against these epitopes could potentially inhibit theinteraction of B7 with its counter-receptor on the T cell. Furthermore,antibodies that cross-reacted with this region on both B7.1 and B7.2molecules would potentially have practical advantages over antibodiesdirected against B7.1 or B7.2 separately.

2. Blockade of the B7/CD28 Interaction

Blocking of the B7/CD28 interaction offers the possibility of inducingspecific immunosuppression, with potential for generating long lastingantigen-specific therapeutic effects. Antibodies or agents thattemporarily prevent this interaction may be useful, specific and safeclinical immunosuppressive agents, with potential for generating longterm antigen-specific therapeutic effects.

Antibodies to either B7.1 or B7.2 have been shown to block T cellactivation, as measured by the inhibition of IL-2 production in vitro(DeBoer, M., P. Parren, J. Dove, F. Ossendorp, G. van der Horst, and J.Reeder (1992), “Functional Characterization of a Novel Anti-B7Monoclonal Antibody,” Eur. Journal of Immunology 22:3071-3075; Azuma,M., H. Yssel, J. H. Phillips, H. Spits, and L. L. Lanier (1993),“Functional Expression of B7/BB1 on Activated T Lymphocytes,” J. Exp.Med. 177:845-850)). However, different antibodies have been shown tovary in their immunosuppressive potency, which may reflect either theiraffinity or epitope specificity. A possible explanation for this mayreside in the ability of some antibodies to block only the binding of B7to CD28, while promoting apoptosis or some other form of negativesignaling through the CTLA-4 receptor in activated T cells. Someantibodies to B7.1 or B7.2 may, in fact, hinder the activity of CTLA-4by cross-reacting with the CTLA-4 binding domain. CTLA-4Ig fusionprotein and anti-CD28 Fabs were shown to have similar effects on thedown regulation of IL-2 production.

In vivo administration of a soluble CTLA-4Ig fusion protein has beenshown to suppress T cell dependent antibody responses in mice (Linsley,P. S., J. L. Greene, P. Tan, J. Bradshaw, J. A. Ledbetter, C. Anasetti,and N. K. Damle (1992), “Coexpression and Functional Cooperation ofCTLA-4 and CD28 on Activated T Lymphocytes,” J. Exp. Med. 176:1595-1604;Lin, H., S. F. Builing, P. S. Linsley, R. O. Wei, C. D. Thompson, and L.A. Turka (1993), “Long-term Acceptance of Major HistocompatibilityComplex Mismatched Cardiac Allografts Induced by CTLA-4-Ig Plus DonorSpecific Transfusion,” J. Exp. Med. 178:1801) and, furthermore, largerdoses were also able to suppress responses to a second immunization,demonstrating the feasibility of this approach for the treatment ofantibody mediated autoimmune disease. In addition, CTLA-4Ig was able toprevent pancreatic islet cell rejection in mice by directly inhibitingthe interaction of T cells and B7.1/B7.2 antigen presenting cells(Lenschow, D. J., G. H. Su, L. A. Zuckerman, N. Nabavi, C. L. Jellis, G.S. Gray, J. Miller, and J. A. Bluestone (1993), “Expression andFunctional Significance of an Additional Ligand for CTLA-4,” Proc. Natl.Acad. Sci., USA 90:11054-11058). In this case, long term donor specifictolerance was achieved.

3. Recombinant Phage Display Technology for Antibody Selection

To date, no monoclonal antibodies which cross-react with both B7.1 andB7.2 have been reported. Furthermore, no monoclonal antibodies which arespecific to B7.1 or B7.2 and which also recognize specific sites on theantigens which are restricted to co-activation receptor CD28 bindinghave been reported. Or alternatively, no monoclonal antibodies which arespecific to B7.1 or B7.2 and which recognize specific sites on theantigens which are exclusive of CTLA-4 receptor binding have beenreported. As discussed supra, such antibodies would potentially behighly desirable as immunosuppressants.

Phage display technology is beginning to replace traditional methods forisolating antibodies generated during the immune response, because amuch greater percentage of the immune repertoire can be assessed than ispossible using traditional methods. This is in part due to PEG fusioninefficiency, chromosomal instability, and the large amount of tissueculture and screening associated with heterohybridoma production. Phagedisplay technology, by contrast, relies on molecular techniques forpotentially capturing the entire repertoire of immunoglobulin genesassociated with the response to a given antigen.

This technique is described by Barbas et al, Proc. Natl. Acad. Sci., USA88:7978-7982 (1991). Essentially, immunoglobulin heavy chain genes arePCR amplified and cloned into a vector containing the gene encoding theminor coat protein of the filamentous phage M13 in such a way that aheavy chain fusion protein is created. The heavy chain fusion protein isincorporated into the M13 phage particle together with the light chaingenes as it assembles. Each recombinant phage contains, within itsgenome, the genes for a different antibody Fab molecule which itdisplays on its surface. Within these libraries, in excess of 10⁶different antibodies can be cloned and displayed. The phage library ispanned on antigen coated microliter wells, non-specific phage are washedoff, and antigen binding phage are eluted. The genome from theantigen-specific clones is isolated and the gene III is excised, so thatantibody can be expressed in soluble Fab form for furthercharacterization. Once a single Fab is selected as a potentialtherapeutic candidate, it may easily be converted to a whole antibody. Apreviously described expression system for converting Fab sequences towhole antibodies is IDEC's mammalian expression vector NEOSPLA. Thisvector contains either human gamma 1 or gamma 4 constant region genes.CHO cells are transfected with the NEOSPLA vectors and afteramplification this vector system has been reported to provide very highexpression levels (>30 pg/cell/day) can be achieved.

4. Primatized PRIMATIZED® Antibodies

Another highly efficient means for generating recombinant antibodies isdisclosed by Newman (1992), Biotechnology 10, 1455-1460. Moreparticularly, this technique results in the generation of PRIMATIZED®antibodies which contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein.

Moreover, this technique is also described in commonly assigned U.S.Ser. No. 08/379,072, filed on Jan. 25, 1995, now U.S. Pat. No.5,658,570, which is a continuation of U.S. Ser. No. 07/912,292, filedJul. 10, 1992, which is a continuation-in-part of U.S. Ser. No.07/856,281, filed Mar. 23, 1992, which is a continuation-in-part of U.S.Ser. No. 07/735,064, filed Jul. 25, 1991. U.S. Pat. No. 5,658,570, andthe parent applications thereof are incorporated by reference in theirentirety herein.

This technique modifies antibodies such that they are not antigenicallyrejected upon administration in humans. This technique relies onimmunization of cynomolgus monkeys with human antigens or receptors.This technique was developed to create high affinity monoclonalantibodies directed to human cell surface antigens.

Identification of macaque antibodies to human B7.1 and B7.2 by screeningof phage display libraries or monkey heterohybridomas obtained using Blymphocytes from B7.1 and/or B7.2 immunized monkeys is also described incommonly assigned U.S. application Ser. No. 08/487,550, filed Jun. 7,1995, incorporated by reference in its entirety herein. Morespecifically, Ser. No. 08/487,550 provides four monoclonal antibodies7B6, 16C10, 7C10 and 20C9 which inhibit the B7:CD28 pathway and therebyfunction as effective immunosuppressants.

Antibodies generated in the manner described by these co-assignedapplications have previously been reported to display human effectorfunction, have reduced immunogenicity, and long serum half-life. Thetechnology relies on the fact that despite the fact that cynomolgusmonkeys are phylogenetically similar to humans, they still recognizemany human proteins as foreign and therefore mount an immune response.Moreover, because the cynomolgus monkeys are phylogenetically close tohumans, the antibodies generated in these monkeys have been discoveredto have a high degree of amino acid homology to those produced inhumans. Indeed, after sequencing macaque immunoglobulin light and heavychain variable region genes, it was found that the sequence of each genefamily was 85-98% homologous to its human counterpart (Newman et al,(1992), Id.). The first antibody generated in this way, an anti-CD4antibody, was 91-92% homologous to the consensus sequence of humanimmunoglobulin framework regions. Newman et al, Biotechnology10:1458-1460 (1992).

Monoclonal antibodies specific to the human B7 antigen have beenpreviously described in the literature. For example, Weyl et al, Hum.Immunol. 31(4), 271-276 (1991) describe epitope mapping of humanmonoclonal antibodies against HLA-B-27 using natural and mutatedantigenic variants. Also, Toubert et al, Clin. Exp. Immunol. 82(1),16-20 (1990) describe epitope mapping of an HLA-B27 monoclonal antibodythat also reacts with a 35-KD bacterial outer membrane protein. Also,Valle et al, Immunol. 69(4), 531-535 (1990) describe a monoclonalantibody of the IgG1 subclass which recognizes the B7 antigen expressedin activated B cells and HTLV-1-transformed T cells. Further, Toubert etal, J. Immunol. 141(7), 2503-9 (1988) describe epitope mapping ofHLA-B27 and HLA-B7 antigens using intradomain recombinants constructedby making hybrid genes between these two alleles in E. coli.

High expression of B7 antigen has been correlated to autoimmune diseasesby some researchers. For example, Ionesco-Tirgoviste et al., Med.Interre 24(1), 11-17 (1986) report increased B7 antigen expression intype 1 insulin-dependent diabetes. Also, the involvement of B7 antigenexpression on dermal dendritic cells obtained from psoriasis patientshas been reported. (Nestle et al, J. Clin. Invest. 94(1), 202-209(1994)).

Further, the inhibition of anti-HLA-B7 alloreactive CTL usingaffinity-purified soluble HLA-B7 has been reported in the literature.(Zavazava et al, Transplantation 51(4), 838-42 (1991)). Further, the useof B7 receptor soluble ligand, CTLA-4-Ig to block B7 activity (See,e.g., Lenschow et al, Science 257, 789, 7955 (1992)) in animal modelsand a B7.1-Ig fusion protein capable of inhibiting B7 has been reported.

Evidence is provided in this disclosure for the identification ofmonoclonal antibodies which recognize specific sites on the B7.1 antigenwhich are restricted to CD28 receptor binding. Furthermore, evidence ispresented herein for the identification of antibodies which recognizesites on the B7.1 antigen which are exclusive of CTLA-4 receptorbinding. Thus, evidence is presented herein to support the existence ofunique antigen binding sites on the human B7.1 (CD80) co-stimulatoryantigen. The sites claimed are identified by anti-B7.1 PRIMATIZED®antibodies and evidence is presented which confirms binding to a site ofinteraction on the B7.1 antigen which is restricted to binding with theco-activation receptor CD28.

SUMMARY AND OBJECTS OF THE INVENTION

An object of the invention is to identify novel antibodies which arespecific to human B7.1 antigen. More specifically, it is an object ofthe invention to identify antibodies which are specific to human B7.1antigen and which are also capable of inhibiting the binding of B7.1 toa CD28 receptor. It is also an object of this invention to identifyantibodies which are specific to human B7.1 antigen and which are notcapable of inhibiting the binding of B 7.1 to a CTLA-4 receptor. Thus,an object of this invention is to identify antibodies which recognizespecific sites on the B7.1 antigen, wherein the recognized sites arerestricted to CD28 receptor binding and which are exclusive of CTLA-4receptor binding.

It is a further object of the invention to identify antibodies which arespecific to human B7.1 antigen and which fail to recognize human B7.2antigen.

It is another object of the invention to identify monoclonal antibodiesand PRIMATIZED® forms thereof which recognize specific sites on thehuman B7.1 antigen and which inhibit IL-2 production and T cellproliferation and which function as effective immunosuppressants. Morespecifically, it is an object of this invention to identify antibodieswhich are specific to B7.1 and which are capable of inhibiting IL-2production.

It is another object of the invention to provide monoclonal antibodiesand PRIMATIZED® forms thereof which inhibit antigen driven responses indonor spleen cell cultures, e.g., antigen specific IgG responses, IL-2production and cell proliferation.

It is another specific object of the invention to identify particularmonoclonal antibodies specific to human B7.1 antigen and PRIMATIZED®forms thereof having advantageous properties, i.e., affinity,immunosuppressive activity, which are useful as therapeutics. Morespecifically, these antibodies and PRIMATIZED® forms thereof are to beused, e.g., as immunosuppressants, i.e., to block antigen driven immuneresponses, to treat autoimmune diseases such as psoriasis, rheumatoidarthritis, systemic erythematosus (SLE), type 1 diabetes mellitus,idiopathic thrombocytopenia purpura (ITP), allergy, inflammatory biledisease, and to prevent organ rejection.

It is another object of the invention to provide pharmaceuticalcompositions containing one or more monoclonal antibodies specific tohuman B7.1 antigen or PRIMATIZED® forms thereof, and a pharmaceuticallyacceptable carrier or excipient. These compositions will be used, e.g.,as immunosuppressants to treat autoimmune diseases, e.g., idiopathicthrombocytopeniapurpura (ITP) and systemic lupus erythematosus (SLE), toblock antigen driven immune responses, and to prevent organ rejection intransplant recipients.

It is another object of the invention to provide novel methods oftherapy by administration of therapeutically effective amounts of one ormore or PRIMATIZED® monoclonal antibodies which specifically bind tohuman B7.1 antigen. Such therapeutic methods are useful for treatment ofdiseases treatable by inhibition of the B7:CD28 pathway, e.g.,autoimmune diseases such as idiopathic thrombocytopenia purpura (ITP),systemic lupus erythematosus (SLE), type 1 diabetes mellitus, psoriasis,rheumatoid arthritis, multiple sclerosis, aplastic anemia, as well asfor preventing rejection in transplantation subjects

It is still another object of the invention to provide transfectants,e.g., CHO cells, which express at least the variable heavy and lightdomains of monoclonal antibodies specific to the human B7.1 antigen.

DEFINITIONS

The following terms are defined so that the invention may be moreclearly understood.

Depleting antibody—an antibody which kills activated B cells or otherantigen presenting cells.

Non-depleting antibody—an antibody which blocks the co-stimulatoryaction of B7 and T cell activating ligands CD28 and CTLA-4. Thus, itanergizes but does not eliminate the antigen presenting cell.

PRIMATIZED® antibody—a recombinant antibody which has been engineered tocontain the variable heavy and light domains of a monkey antibody, inparticular, a cynomolgus monkey antibody, and which contains humanconstant domain sequences, preferably the human immunoglobulin gamma 1or gamma 4 constant domain (or PE variant). The preparation of suchantibodies is described in Newman et al, (1992), “Primatization ofRecombinant Antibodies for Immunotherapy of Human Diseases: AMacaque/Human Chimeric Antibody Against Human CDH”, Biotechnology10:1458-1460; also in commonly assigned U.S. Ser. No. 08/379,072, nowU.S. Pat. No. 5,658,570, both of which are incorporated by reference intheir entirety herein. These antibodies have been reported to exhibit ahigh degree of homology to human antibodies, i.e., 85-98%, display humaneffector functions, have reduced immunogenicity, and may exhibit highaffinity to human antigens.

B7 antigens—B7 antigens in this application include, e.g., human B7,B7.1 and B7.2 antigens. These antigens bind to CD28 and/or CTLA-4. Theseantigens have a co-stimulatory role in T cell activation. Also, these B7antigens all contain extracellular immunoglobulin superfamily V andC-like domains, a hydrophobic transmembrane region and a cytoplasmictail (See, Freeman et al, Science 262:909, (1993)), and are heavilyglycosylated.

Anti-B7 antibodies—Antibodies, preferably monkey monoclonal antibodiesor PRIMATIZED® forms thereof, which specifically bind human B7 antigens,e.g., human B7.1 and/or B7.2 antigen with a sufficient affinity to blockthe B7:CD28 interaction, but do not block the B7/CTLA-4 receptorinteraction and thereby induce immunosuppression.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the pMS vector used to screen recombinant immunoglobulinlibraries produced against B7 displayed on the surface of filamentousphage which contains primers based on macaque immunoglobulin sequences.

FIG. 2 depicts the NEOSPLA® expression vector used to express thesubject PRIMATIZED® antibodies specific to human B7.1 antigen.

FIG. 3A depicts the amino acid and nucleic acid sequence of aPRIMATIZED® form of the light chain of 7C10 (SEQ ID NOS: 1-2).

FIGS. 3B and 3C depict the amino acid and nucleic acid sequence of aPRIMATIZED® form of the light chain of 7C10 (SEQ ID NOS: 3-4).

FIG. 4A depicts the amino acid and nucleic acid sequence of aPRIMATIZED® form of the light chain of 7B6 (SEQ ID NOS: 5-6).

FIGS. 4B and 4C depict the amino acid and nucleic acid sequence of aPRIMATIZED® form of the heavy chain of 7B6 (SEQ ID NOS: 7-8).

FIG. 5A depicts the amino acid and nucleic acid sequence of aPRIMATIZED® light chain 16C10 (SEQ ID NOS: 9-10).

FIGS. 5B and 5C depict the amino acid and nucleic acid sequence of aPRIMATIZED® heavy chain 16C10 (SEQ ID NOS: 11-12).

FIG. 6 depicts the inability of P16C10 to block CTLA-4Ig-Biotin bindingto B7.1 transfected CHO cells.

FIG. 7 depicts the inability of CTLA-4Ig to block P16C10-Biotin bindingto B7.1 transfected CHO cells.

FIG. 8 depicts that BB-1 completely blocks binding of CTLA-4Ig-Biotin toB7.1 transfected CHO cells and further depicts the inability of BB-1 tosignificantly affect P16C10-Biotin binding to B7.1 transfected CHOcells.

FIG. 9 depicts that CTLA-4Ig-Biotin is effectively blocked by all B7.1inhibitors except P16C10.

FIG. 10 depicts the ability of P16C10 to block binding of theCD28/B7-1Ig interaction. Data shown are averages of values obtained fromfour separate experiments.

FIG. 11 depicts production of IL-2 in cultures of purified normal humanCD4+ lymphocytes when stimulated with sub-optimal amounts of immobilizedanti-CD3 antibody and B7-1 (CD80) on latex microbeads. L307.4 is acommercially available murine antibody (BID Pharmingen) that bindsspecifically to human CD80 and neutralizes CD28:CD80 functionalinteractions. CTLA-4Ig is a soluble receptor fusion protein thatspecifically blocks CD80 and CD86 binding to CD28 receptors on T cells.IDEC-114 is a PRIMATIZED® monoclonal antibody that specifically binds toboth soluble and membrane forms of the CD80 antigen but does notrecognize CTLA-4 or B7-2 antigens. The ratio of anti-CD3 to B7Ig used inthe cultures to stimulate T cells was 1:10 (w/w).

FIG. 12 depicts uptake of H3-Thymidine in cultures of purified normalhuman CD4+ lymphocytes when stimulated with sub-optimal amounts ofimmobilized anti-CD3 antibody and B7-1 (CD80) on latex microbeads.L307.4 is a commercially available murine antibody (B/D Pharmingen) thatbinds specifically to human CD80 and neutralizes CD28:CD80 functionalinteractions. CTLA-4Ig is a soluble receptor fusion protein thatspecifically blocks CD80 and CD86 binding to CD28 receptors on T cells.IDEC-114 is a PRIMATIZED® monoclonal antibody that specifically binds toboth soluble and membrane forms of the CD80 antigen but does notrecognize CTLA-4 or B7-2 antigens.

FIG. 13 depicts production of TH2 cytokine IL-10 in cultures of purifiednormal human CD4+ lymphocytes when stimulated with sub-optimal amountsof immobilized anti-CD3 antibody and B7-1 (CD80) on latex microbeads.Inhibition of L-10 production by L307.4 anti-CD80 and CTLA-4Ig fusionprotein was compared at 0.1, 1, and 10 μg/mL.

FIG. 14 depicts inhibition of IL-2 cytokine production by CTLA-4Ig andIDEC-114 in cultures of purified human CD4+ T cells. T cells wereco-stimulated with anti-CD3 and B7Ig coated latex microbeads with ananti-CD3/B7 ratio (w/w) of 8:1. IL-2 was determined by growth and uptakeof Thymidine by the IL-2 dependent cell line CTLL-2.

DETAILED DESCRIPTION OF THE INVENTION

As described above, the present invention relates to the identificationof monoclonal antibodies or PRIMATIZED® forms thereof which are specificto human B7.1 antigen and which are capable of inhibiting the binding ofB7.1 to a CD28 receptor and which are not capable of inhibiting thebinding of B7.1 to a CTLA-4 receptor. Blocking of the primary activationsite between CD28 and B7.1 (CD80) with the identified antibodies whileallowing the combined antagonistic effect on positive co-stimulationwith an agnostic effect on negative signaling will be a usefultherapeutic approach for intervening in relapsed forms of autoimmunedisease. The functional activity of the identified antibodies is definedby blocking the production of the T cell stimulatory cytokine IL-2.Identified antibodies have demonstrated the ability to block theproduction of IL-2 in excess of 50%, in spite of the existence of asecond actuating ligand B7.2, suggesting an alternate mechanism ofaction exists which is not typical of the observed effects of otheranti-B7.1 antibodies defined in the literature.

Manufacture of novel monkey monoclonal antibodies which specificallybind human B7.1 and/or human B7.2 antigen, as well as PRIMATIZED®antibodies derived therefrom is described in co-pending U.S. applicationSer. No. 08/487,550, and as set forth herein. These antibodies possesshigh affinity to human B7.1 and/or B7.2 and therefore may be used asimmunosuppressants which inhibit the B7:CD86 pathway.

Preparation of monkey monoclonal antibodies will preferably be effectedby screening of phage display libraries or by preparation of monkeyheterohybridomas using B lymphocytes obtained from B7 (e.g., human B7.1and/or B7.2) immunized monkeys.

As noted, the first method for generating anti-B7 antibodies involvesrecombinant phage display technology. This technique is generallydescribed supra.

Essentially, this will comprise synthesis of recombinant immunoglobulinlibraries against B7 antigen displayed on the surface of filamentousphage and selection of phage which secrete antibodies having highaffinity to B7.1 and/or B7.2 antigen. As noted supra, preferablyantibodies will be selected which bind to both human B7.1 and B7.2. Toeffect such methodology, the present inventors have created a uniquelibrary for monkey libraries which reduces the possibility ofrecombination and improves stability. This vector, PMS, is described indetail infra, and is shown in FIG. 1.

Essentially, to adopt phage display for use with macaque libraries, thisvector contains specific primers for PCR amplifying monkeyimmunoglobulin genes. These primers are based on macaque sequencesobtained while developing the PRIMATIZED® technology and databasescontaining human sequences.

Suitable primers are disclosed in commonly assigned U.S. Pat. No.5,658,570.

The second method involves the immunization of monkeys, i.e., macaques,against human B7 antigen, preferably against human B7.1 and B7.2antigen. The inherent advantage of macaques for generation of monoclonalantibodies is discussed supra. In particular, such monkeys, i.e.,cynomolgus monkeys, may be immunized against human antigens orreceptors. Moreover, the resultant antibodies may be used to makePRIMATIZED® antibodies according to the methodology of Newman et al,Biotechnology 10, 1455-1460 (1992), and Newman et al, commonly assignedU.S. Ser. No. U.S. Pat. No. 5,658,570, which are incorporated byreference in their entirety.

The significant advantage of antibodies obtained from cynomolgus monkeysis that these monkeys recognize many human proteins as foreign andthereby provide for the formation of antibodies, some with high affinityto desired human antigens, e.g., human surface proteins and cellreceptors. Moreover, because they are phylogenetically close to humans,the resultant antibodies exhibit a high degree of amino acid homology tothose produced in humans. As noted above, after sequencing macaqueimmunoglobulin light and heavy variable region genes, it was found thatthe sequence of each gene family was 85-88% homologous to its humancounterpart (Newman et al, (1992), Id.).

Essentially, cynomolgus macaque monkeys are administered human B7antigen, e.g., human B7.1 and/or human B7.2 antigen, B cells areisolated therefrom, e.g., lymph node biopsies are taken from theanimals, and B lymphocytes are then fused with KH6/B5 (mouse×human)heteromyeloma cells using polyethylene glycol (PEG). Heterohybridomassecreting antibodies which bind human B7 antigen, e.g., human B7.1and/or human B7.2 antigen, are then identified.

Antibodies which bind to both B7.1 and B7.2 are desirable because suchantibodies potentially may be used to inhibit the interaction of B7.1and B7.2, as well as B7 with their counter-receptors, i.e., human CTLA-4and CD28. Antibodies against these epitopes may inhibit the interactionof both human B7.1 and human B7.2 with their counter receptors on the Tcell. This may potentially provide synergistic effects.

However, antibodies which bind to only one of human B7 antigen, B7.1antigen or B7.2 antigen, are also highly desirable because of theco-involvement of these molecules in T cell activation, clonal expansionlymphokine (IL-2) secretion, and responsiveness to antigen. Given thatboth human B7.1 and B7.2 bind to human CTLA-4 and CD28, it is probablethat there is at least one common or homologous region (perhaps a sharedconformational epitope or epitopes) to which macaque antibodies maypotentially be raised.

The disclosed invention involves the use of an animal which is primed toproduce a particular antibody. Animals which are useful for such aprocess include, but are not limited to, the following: mice, rats,guinea pigs, hamsters, monkeys, pigs, goats and rabbits.

A preferred means of generating human antibodies using SCID mice isdisclosed in commonly-owned, co-pending U.S. patent application Ser. No.08/488,376.

The present inventors elected to immunize macaques against human B7.1antigen using recombinant soluble B7.1 antigen produced in CHO cells andpurified by affinity chromatography using a L307.4-SEPHAROSE® affinitycolumn. However, the particular source of human B7 antigen, human B7.1antigen or human B7.2 antigen is not critical, provided that it is ofsufficient purity to result in a specific antibody response to theparticular administered B7 antigen and potentially to other B7 antigens.

The human B7 antigen, human B7.1 antigen (also called CD80) and humanB7.2 antigen (also called CD86) genes have been cloned, and sequenced,and therefore may readily be manufactured by recombinant methods.

Preferably, the administered human B7 antigen, human B7.1 antigen and/orhuman B7.2 antigen will be administered in soluble form, e.g., byexpression of a B7, B7.1 or B7.2 gene which has its transmembrane andcytoplasmic domains removed, thereby leaving only the extracellularportion, i.e., the extracellular superfamily V and C-like domains. (See,e.g., Grumet et al, Hum. Immunol. 40(3), p. 228-234 (1994), whichteaches expression of a soluble form of human B7, which is incorporatedby reference in its entirety herein)).

The macaques will be immunized with the B7, B7.1 and/or B7.2 antigen,preferably a soluble form thereof, under conditions which result in theproduction of antibodies specific thereto. Preferably, the soluble humanB7, B7.1 or B7.2 antigen will be administered in combination with anadjuvant, e.g., Complete Freund's Adjuvant (CFA), Alum, Saponin, orother known adjuvants, as well as combinations thereof. In general, thiswill require repeated immunization, e.g., by repeated injection, overseveral months. For example, administration of soluble B7.1 antigen waseffected in adjuvant, with booster immunizations, over a 3 to 4 monthperiod, with resultant production of serum containing antibodies whichbound human B7.1 antigen.

After immunization B cells are collected, e.g., by lymph node biopsiestaken from the immunized animals and B lymphocytes fused with KH6/B5(mouse×human) heteromyeloma cells using polyethylene glycol. Methods forpreparation of such heteromyelomas are known and may be found in U.S.Pat. No. 5,658,570, by Newman et al.

Heterohybridomas which secrete antibodies which bind human B7, B7.1and/or B7.2 are then identified. This may be effected by knowntechniques. For example, this may be determined by ELISA orradioimmunoassay using enzyme or radionucleotide labelled human B7, B7.1and/or B7.2 antigen.

Cell lines which secrete antibodies having the desired specificity tohuman B7, B7.1 and/or B7.2 antigen are then subcloned to monoclonality.

In the present invention, the inventors screened purified antibodies fortheir ability to bind to soluble B7.1 antigen coated plates in an ELISAassay, antigen positive B cells, and CHO transfectomas which expresshuman B7.1 antigen on their cell surface. In addition, the antibodieswere screened for their ability to block B cell/T cell interactions asmeasured by IL-2 production and tritiated thymidine uptake in a mixedlymphocyte reaction (MLR), with B7 binding being detected using¹²⁵I-radiolabeled soluble B7.1 (SB7.1).

Also, affinity purified antibodies from macaques were tested for theirreactivity against CHO transfectants which expressed B7.1/Ig fusionproteins, and against CHO cells which produced human B7.2 antigen. Theseresults indicated that the B7.1 immune sera bound to the B7.2transfectomas. Binding of antibodies to B7.2 antigen may be confirmedusing soluble B7.2-Ig reagents. As discussed in the examples, this maybe effected by producing and purifying B7.2-Ig from CHO transfectomas insufficient quantities to prepare a B7.2-Ig-SEPHAROSE® affinity column.Those antibodies which cross-react with B7.2 will bind theB7.2-Ig-SEPHAROSE® column.

Cell lines which express antibodies which specifically bind to human B7antigen, B7.1 antigen and/or B7.2 antigen are then used to clonevariable domain sequences for the manufacture of PRIMATIZED® antibodiesessentially as described in Newman et al (1992), Id. and Newman et al,U.S. Ser. No. 379,072, filed Jan. 25, 1995, both of which areincorporated by reference herein. Essentially, this entails extractionof RNA therefrom, conversion to cDNA, and amplification thereof by PCRusing Ig specific primers. Suitable primers are described in Newman etal, 1992, Id. and in U.S. Ser. No. 379,072. (See, in particular, FIG. 1of U.S. Ser. No. 379,072).

The cloned monkey variable genes are then inserted into an expressionvector which contains human heavy and light chain constant region genes.Preferably, this is effected using a proprietary expression vector ofIDEC, Inc., referred to as NEOSPLA®. This vector is shown in FIG. 2 andcontains the cytomegalovirus promoter/enhancer, the mouse beta globinmajor promoter, the SV40 origin of replication, the bovine growthhormone polyadenylation sequence, neomycin phosphotransferase exon 1 andexon 2, human immunoglobulin kappa or lambda constant region, thedihydrofolate reductase gene, the human immunoglobulin gamma 1 or gamma4 PE constant region and leader sequence. This vector has been found toresult in very high level expression of PRIMATIZED® antibodies uponincorporation of monkey variable region genes, transfection in CHOcells, followed by selection in G418 containing medium and methotrexateamplification.

For example, this expression system has been previously disclosed toresult in PRIMATIZED® antibodies having high avidity (Kd≦10⁻¹⁰ M)against CD4 and other human cell surface receptors. Moreover, theantibodies have been found to exhibit the same affinity, specificity andfunctional activity as the original monkey antibody. This vector systemis substantially disclosed in commonly assigned U.S. Pat. No. 5,658,570,incorporated by reference herein, as well as U.S. Ser. No. 08/149,099,now U.S. Pat. No. 5,736,137, also incorporated by reference in itsentirety herein. This system provides for high expression levels,i.e., >30 pg/cell/day.

Using the techniques described supra, and in commonly assigned U.S. Pat.No. 5,658,570, the present inventors have cloned the variable domains of7C10, 7B6 and 16C10, and provide the amino acid and nucleic acidsequences of PRIMATIZED® forms of the 7C10 light chain, 7C10 heavychain, 7B6 light chain, 7B6 heavy chain, 16C10 light chain and 16C10heavy chain. These amino acid and nucleic acid sequences may be found inFIGS. 3A (SEQ ID NOS: 1-2), 3B-3C (SEQ ID NOS: 3-4), 4A (SEQ ID NOS:5-6), 4B-4C (SEQ ID NOS: 7-8), 5A (SEQ ID NOS: 9-10) and 5B-5C (SEQ IDNOS: 11-12). The DNA and amino acid sequence for the human gamma 1,gamma 4 constant domain may be found in U.S. Pat. No. 5,658,570.

As discussed infra, the subject inventors have selected four leadcandidate monkey monoclonal antibodies which specifically bind the B7.1antigen. These monkey monoclonal antibodies are referred to herein as7B6, 16C10, 7C10 and 20C9.

As discussed in greater detail infra, these antibodies were evaluatedfor their ability to block B cell/T cell interactions as measured byIL-2 production and tritiated thymidine uptake in a mixed lymphocytereaction for T cell binding experiments for T cell binding, human buffycoat peripheral blood lymphocytes were cultured for 3-6 days in thepresence of PHA stimulator. B7 binding was radioassayed using¹²⁵I-radiolabeled soluble B7.1. The observed results indicate that allof these antibodies bind B7.1 antigen with high affinity and effectivelyblock B cell/T cell interactions as evidenced by reduced IL-2 productionand reduced proliferation of mixed lymphocyte cultures.

The properties of these particular monkey monoclonal antibodies aresummarized below:

-   -   1. Scatchard analysis showed that the apparent affinity        constants (Kd) for the monkey antibodies binding to B7-Ig coated        plates were approximated to be:        -   a: 7C10: 6.2×10⁻⁹M        -   b: 16C10: 8.1×10⁻⁹M        -   c: 7B6: 10.7×10⁻⁹M        -   d: 20C9: 16.8×10⁻⁹M    -   2. The antibodies were tested in vitro in a mixed lymphocyte        reaction assay (MLR). The MLR showed that all 4 anti-B7.1        antibodies inhibit IL-2 production to different extents as shown        by the following IC₅₀ values:        -   a: 7B6: 5.0 μg/M        -   b: 16C10: <0.1 μg/M        -   c: 20C9: 2.0 μg/M        -   d: 7C10: 5.0 μg/M    -   3. The monkey anti-B7.1 antibodies were tested for their ability        to bind B7 on human peripheral blood lymphocytes (PBL). FACS        analysis showed that all 4 monkey antibodies tested positive.    -   4. Monkey antibodies 16C10, 7B6, 7C10 and 20C9 were tested for        C1q binding by FACS analysis. Results showed 7C10 monkey Ig had        strong human C1q binding after incubating with B7.1        CHO-transfected cells. 16C10 was positive, while 20C9 and 7B6        monkey antibodies were negative.    -   5. To select an animal model for path-tox studies, the monkey        antibodies were tested with animal blood from different species.        It was determined that the monkey anti-B7.1 antibodies        cross-reacted with human, chimpanzee.

Based on these properties, it would appear that three monkey monoclonalantibodies possess the most advantageous properties, 16C10, 7C10 and20C9, with 16C10 and 7C10 being somewhat better than 20C9.

As previously noted, the subject PRIMATIZED® antibodies will preferablycontain either the human immunoglobulin gamma 1 or gamma 4 constantregion, with gamma 4 preferably mutated at two positions to create gamma4 PE. The gamma 4 PE mutant contains two mutations, a glutamic acid inthe CH2 region introduced to eliminate residual FCR binding, and aproline substitution in the hinge region, intended to enhance thestability of the heavy chain disulfide bond interaction. (See, Alegre etal, J. Immunol. 148, 3461-3468 (1992); and Angel et al, Mol. Immunol.30, 105-158 (1993), both of which are incorporated by reference herein).

As discussed supra, these primatized antibodies are preferably expressedusing the NEOSPLA expression vector shown in FIG. 2 which issubstantially described in commonly assigned U.S. Pat. No. 5,658,570,and U.S. Pat. No. 5,736,137, incorporated by reference herein in itsentirety.

Whether the subject PRIMATIZED® antibodies contain the gamma 1, gamma 4or gamma 4 PE constant region largely depends on the particular diseasetarget. Preferably, depleting and non-depleting PRIMATIZED® IgG1 andIgG4 antibodies are created and tested against specific disease targets.

Given the described binding and functional properties of the subjectmonkey monoclonal antibodies, these anti-B7.1 monoclonal antibodies andPRIMATIZED® forms thereof should be well suited as therapeutic agentsfor blocking the B7:CD28 interaction thereby providing forimmunosuppression. In particular, given their high affinity to B7.1antigen and ability to block B cell/T cell interactions as measured byIL-2 production and tritiated thymidine uptake in mixed lymphocyteculture as well as their ability to effectively inhibit antigen drivenresponses in donor spleen cell cultures as shown by reduced antigenspecific IgG responses, IL-2 production and cell proliferation, thesemonkey monoclonal antibodies and PRIMATIZED® forms thereof shouldfunction as effective immunosuppressants which modulate the B7:CD28pathway. This is significant for the treatment of many diseases whereinimmunosuppression is therapeutically desirable, e.g., autoimmunediseases, to inhibit undesirable antigen specific IgG responses, andalso for prevention of organ rejection and graft-versus-host disease.Essentially, the subject antibodies will be useful in treating anydisease wherein suppression of the B7:CD28 pathway is therapeuticallydesirable.

Key therapeutic indications for the subject anti-B7.1 antibodiesinclude, by way of example, autoimmune diseases such as idiopathicthrombocytopenia purpura (ITP), systemic lupus erythematosus (SLE), type1 diabetes mellitus, multiple sclerosis, aplastic anemia, psoriasis,allergy, inflammatory bile disease and rheumatoid arthritis.

Another significant therapeutic indication of the subject anti-B7.1antibodies is for prevention of graft-versus-host-disease (GVHD) duringorgan transplant and bone marrow transplant (BMT). The subjectantibodies may be used to induce host tolerance to donor-specificalloantigens and thereby facilitate engraftment and reduce the incidenceof graft rejection. It has been shown in a murine model of allogeneiccardiac transplantation that intravenous administration of CTLA4-Ig canresult in immunosuppression or even induction of tolerance toalloantigen. (Lin et al, J. Exp. Med. 178:1801, 1993; Torka et al, Proc.Natl. Acad. Sci., USA 89:11102, 1992). It is expected that the subjectPRIMATIZED® anti-B7.1 antibodies will exhibit similar or greateractivity.

Antibodies produced in the manner described above, or by equivalenttechniques, can be purified by a combination of affinity and sizeexclusion chromatography for characterization in functional biologicalassays. These assays include determination of specificity and bindingaffinity as well as effector function associated with the expressedisotype, e.g., ADCC, or complement fixation. Such antibodies may be usedas passive or active therapeutic agents against a number of humandiseases, including B cell lymphoma, infectious diseases including viraldiseases such as HIV/AIDS, autoimmune and inflammatory diseases, andtransplantation. The antibodies can be used either in their native form,or as part of an antibody/chelate, antibody/drug or antibody/toxincomplex. Additionally, whole antibodies or antibody fragments (Fab₂,Fab, Fv) may be used as imaging reagents or as potential vaccines orimmunogens in active immunotherapy for the generation of anti-idiotypicresponses.

The amount of antibody useful to produce a therapeutic effect can bedetermined by standard techniques well known to those of ordinary skillin the art. The antibodies will generally be provided by standardtechnique within a pharmaceutically acceptable buffer, and may beadministered by any desired route. Because of the efficacy of thepresently claimed antibodies and their tolerance by humans it ispossible to administer these antibodies repetitively in order to combatvarious diseases or disease states within a human.

The anti-B7.1 antibodies (or fragments thereof) of this invention areuseful for inducing immunosuppression, i.e., inducing a suppression of ahuman's or animal's immune system. This invention therefore relates to amethod of prophylactically or therapeutically inducing immunosuppressionin a human or other animal in need thereof by administering aneffective, non-toxic amount of such an antibody of this invention tosuch human or other animal.

The ability of the compounds of this invention to induceimmunosuppression has been demonstrated in standard tests used for thispurpose, for example, a mixed lymphocyte reaction test or a testmeasuring inhibition of T-cell proliferation measured by thymidineuptake.

For example, in vitro assays were conducted that measured cell growthand activating cytokines produced in response to co-stimulatory signalsthat activate CD4+ T cells. The production and secretion of thesecytokines occurs naturally in T cells under conditions where a primaryand secondary signal is generated through interactions between T cellsand antigen presenting cells. Normally a primary signal is initiatedthrough interaction of a antigen specific T cell receptor and MHC ClassII molecules bearing the specific antigen on antigen presenting cells. Asecondary or co-stimulatory signal is required to obtain maximalactivation of T cells. Several T cell co-stimulatory receptors have beenidentified that drive the production of various cytokines, andup-regulate other cell surface receptors that function in growth anddifferentiation of T cells and hematopoietic accessory cells. Some ofthe known signaling T cell co-stimulatory receptors are CD28, CD11, CD54and CD40L. Sustained adhesion and prolonged interactions through thesecell surface molecules result in secretion of IL-2 and various secondaryinflammatory cytokines that control numerous immuno-regulatoryfunctions. The study of T cell interactions can be complex due to thepresence of numerous accessory cell types capable of mediating redundantor interdependent co-stimulatory effects.

The CD28/B7 receptor ligand interaction is considered to be the keysecondary response element between antigen presenting cells and CD4+helper T cells in the immune response cascade. After a primary signal isgenerated between antigen specific T cell receptors and antigen/MHCclass II complexes, two types of B7 molecules, B7-1 (CD80) and B7-2,(CD86) are up-regulated and establish a membrane signaling event throughbinding to CD28 receptor. These signals drive the gene expression ofvarious cytokines beginning with the production of IL-2. The detectionof secreted IL-2, cell proliferation and various cell surface activationmarkers including the receptor for IL-2 are clear indicators thatco-stimulation has occurred and cells are beginning to divide anddifferentiate to maturity. T cells may be influenced or driven downdifferent maturation pathways depending on many complex internal andexternal factors through mechanisms that are poorly understood. TheCD28/B7 interaction was first identified as an adhesion event when a B7specific antibody was identified that blocked adhesion between B and Tcell types. CD28 is known to affect in vivo immune responses byfunctioning both as a cell adhesion molecule linking B and T lymphocytesand as the surface component of a novel signal transduction pathway(June et al. 1990, Immunology Today, 11: 211-216). As a result, severalmonoclonal antibodies that recognize either CD28 or B7 are capable ofblocking both adhesion and signaling events. Blocking of either eventwould lead directly or indirectly to reduced signaling through the CD28receptor and would result in reduced IL-2 production, proliferation andthe appearance of secondary cytokines.

More specifically, the present inventors have isolated certain novelantibodies, the activity of which apparently does not involve directlyblocking of signal transduction as demonstrated through the use ofCTLA-4Ig, a soluble receptor fusion protein that co-recognizes both B7receptors. Evidence is provided herein that a PRIMATIZED® antibodyaccording to the invention, referred to as IDEC-114 blocks adhesion ofantigen presenting cells to T cells thereby blocking an upstream eventprior to signaling that under certain conditions, possibly related to B7receptor density, is capable of influencing T cell activation. Evidenceis provided through use of an in vitro assay that establishes distinctdifferences between the mechanism of action of IDEC-114 and otheranti-CD80 antibodies as well as CTLA-4Ig. The in vitro assay employed inthese experiments was designed to reduce the number of complexinteractions provided by accessory cells, by using a purified CD4+ Tcell population and replacing accessory cells with a non-cellularco-stimulatory system. This cell activating system obviates the need forantigen presenting cells by using latex micospheres containingimmobilized antibody to the CD3 antigen to deliver a suboptimal primarysignal to the T cell. This system when presented along with B7 (CD80)co-stimulatory ligand provides a very potent signal through the CD28receptor that initiates gene expression resulting in production of IL-2,T cell growth and other pro-inflammatory cytokines.

The fact that the antibodies of this invention have utility in inducingimmunosuppression indicates that they should be useful in the treatmentor prevention of resistance to or rejection of transplanted organs ortissues (e.g., kidney, heart, lung, bone marrow, skin, cornea, etc.);the treatment or prevention of autoimmune, inflammatory, proliferativeand hyperproliferative diseases, and of cutaneous manifestations ofimmunologically medicated diseases (e.g., rheumatoid arthritis, lupuserythematosus, systemic lupus erythematosus, Hashimotos thyroiditis,multiple sclerosis, myasthenia gravis, type 1 diabetes, uveitis,nephrotic syndrome, psoriasis, atopical dermatitis, contact dermatitisand further eczematous dermatitides, seborrheic dermatitis, Lichenplanus, Pemplugus, bullous pemphigus, Epidermolysis bullosa, urticaria,angioedemas, vasculitides, erythema, cutaneous eosinophilias, Alopeciagreata, etc.); the treatment of reversible obstructive airways disease,intestinal inflammations and allergies (e.g., inflammatory bile disease,Coeliac disease, proctitis, eosinophilia gastroenteritis, mastocytosis,Crohn's disease and ulcerative colitis), food-related allergies (e.g.,migraine, rhinitis and eczema), and other types of allergies.

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of antibody would be forthe purpose of inducing immunosuppression. Generally, however, aneffective dosage will be in the range of about 0.05 to 100 milligramsper kilogram body weight per day.

The antibodies (or fragments thereof) of this invention should also beuseful for treating tumors in a mammal. More specifically, they shouldbe useful for reducing tumor size, inhibiting tumor growth and/orprolonging the survival time of tumor-bearing animals. Accordingly, thisinvention also relates to a method of treating tumors in a human orother animal by administering to such human or animal an effective,non-toxic amount of an antibody. One skilled in the art would be able,by routine experimentation, to determine what an effective, non-toxicamount of anti-B7 antibody would be for the purpose of treatingcarcinogenic tumors. Generally, however, an effective dosage is expectedto be in the range of about 0.05 to 100 milligrams per kilogram bodyweight per day.

The antibodies of the invention may be administered to a human or otheranimal in accordance with the aforementioned methods of treatment in anamount sufficient to produce such effect to a therapeutic orprophylactic degree. Such antibodies of the invention can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The route of administration of the antibody (or fragment thereof of theinvention may be oral, parenteral, by inhalation or topical. The termparenteral as used herein includes intravenous, intraperitoneal,intramuscular, subcutaneous, rectal or vaginal administration. Thesubcutaneous and intramuscular forms of parenteral administration aregenerally preferred.

The daily parenteral and oral dosage regimens for employing compounds ofthe invention to prophylactically or therapeutically induceimmunosuppression, or to therapeutically treat carcinogenic tumors willgenerally be in the range of about 0.05 to 100, but preferably about 0.5to 10, milligrams per kilogram body weight per day.

The antibodies of the invention may also be administered by inhalation.By “inhalation” is meant intranasal and oral inhalation administration.Appropriate dosage forms for such administration, such as an aerosolformulation or a metered dose inhaler, may be prepared by conventionaltechniques. The preferred dosage amount of a compound of the inventionto be employed is generally within the range of about 10 to 100milligrams.

The antibodies of the invention may also be administered topically. Bytopical administration is meant non-systemic administration and includesthe application of an antibody (or fragment thereof) compound of theinvention externally to the epidermis, to the buccal cavity andinstillation of such an antibody into the ear, eye and nose, and whereit does not significantly enter the blood stream. By systemicadministration is meant oral, intravenous, intraperitoneal andintramuscular administration. The amount of an antibody required fortherapeutic or prophylactic effect will, of course, vary with theantibody chosen, the nature and severity of the condition being treatedand the animal undergoing treatment, and is ultimately at the discretionof the physician. A suitable topical dose of an antibody of theinvention will generally be within the range of about 1 to 100milligrams per kilogram body weight daily.

Formulations

While it is possible for an antibody or fragment thereof to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w butpreferably not in excess of 5% w/w and more preferably from 0.1% to 1%w/w of the formulation.

The topical formulations of the present invention, comprise an activeingredient together with one or more acceptable carrier(s) therefor andoptionally any other therapeutic ingredients(s). The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at90°-100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogols. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

The subject anti-B7.1 antibodies or fragments thereof may also beadministered in combination with other moieties which modulate theB7:CD28 pathway. Such moieties include, by way of example, cytokinessuch as IL-7 and IL-10, CTLA4-Ig, soluble CTLA-4 and anti-CD28antibodies and fragments thereof. Also, the subject antibodies may beadministered in combination with other immunosuppressants. Suchimmunosuppressants include small molecules such as cyclosporin A (CSA)and FK506; monoclonal antibodies such as anti-tumor necrosis factor a(anti-TNFa), anti-CD54, anti-CD11, anti-CD11a, and anti-IL-1; and, othersoluble receptors such as rTNFa and rIL-1.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an antibody or fragmentthereof of the invention will be determined by the nature and extent ofthe condition being treated, the form, route and site of administration,and the particular animal being treated, and that such optimums can bedetermined by conventional techniques. It will also be appreciated byone of skill in the art that the optimal course of treatment, i.e., thenumber of doses of an antibody or fragment thereof of the inventiongiven per day for a defined number of days, can be ascertained by thoseskilled in the art using conventional course of treatment determinationtests.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following formulations are, therefore, to beconstrued as merely illustrative embodiments and not a limitation of thescope of the present invention in any way.

Capsule Composition

A pharmaceutical composition of this invention in the form of a capsuleis prepared by filling a standard two-piece hard gelatin capsule with 50mg. of an antibody or fragment thereof of the invention, in powderedform, 100 mg. of lactose, 32 mg. of talc and 8 mg. of magnesiumstearate.

Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by injection is prepared by stirring 1.5% by weight of anantibody or fragment thereof of the invention in 10% by volume propyleneglycol and water. The solution is sterilized by filtration.

Ointment Composition

Antibody or fragment thereof of the invention 1.0 g.

White soft paraffin to 100.0 g.

The antibody or fragment thereof of the invention is dispersed in asmall volume of the vehicle to produce a smooth, homogeneous product.Collapsible metal tubes are then filled with the dispersion.

Topical Cream Composition

Antibody or fragment thereof of the invention 1.0 g.

Polawax GP 200 20.0 g.

Lanolin Anhydrous 2.0 g.

White Beeswax 2.5 g.

Methyl hydroxybenzoate 0.1 g.

Distilled Water to 100.0 g.

The polawax, beeswax and lanolin are heated together at 60° C. Asolution of methyl hydroxybenzoate is added and homogenization isachieved using high speed stirring. The temperature is then allowed tofall to 50° C. The antibody or fragment thereof of the invention is thenadded and dispersed throughout, and the composition is allowed to coolwith slow speed stirring.

Topical Lotion Composition

Antibody or fragment thereof of the invention 1.0 g.

Sorbitan Monolaurate 0.6 g.

Polysorbate 20 0.6 g.

Cetostearyl Alcohol 1.2 g.

Glycerin 6.0 g.

Methyl Hydroxybenzoate 0.2 g.

Purified Water B. P. to 100-00 ml. (B.P.=British Pharmacopeia)

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml. of thewater at 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenized, allowed to cool with continuousstirring and the antibody or fragment thereof of the invention is addedas a suspension in the remaining water. The whole suspension is stirreduntil homogenized.

Eye Drop Composition

Antibody or fragment thereof of the invention 0.5 g.

Methyl Hydroxybenzoate 0.01 g.

Propyl Hydroxybenzoate 0.04 g.

Purified Water B. P. to 100-00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml. purifiedwater at 75° C. and the resulting solution is allowed to cool. Theantibody or fragment thereof of the invention is then added, and thesolution is sterilized by filtration through a membrane filter (0.022 μmpore size), and packed aseptically into suitable sterile containers.

Composition for Administration by Inhalation

For an aerosol container with a capacity of 15-20 ml: mix 10 mg. of anantibody or fragment thereof of the invention with 0.2-0.5% of alubricating agent, such as polysorbate 85 or oleic acid, and dispersesuch mixture in a propellant, such as freon, preferably in a combinationof (1,2 dichlorotetrafluoroethane) and difluorochloromethane and putinto an appropriate aerosol container adapted for either intranasal ororal inhalation administration.

Composition for Administration by Inhalation

For an aerosol container with a capacity of 15-20 ml: dissolve 10 mg. ofan antibody or fragment thereof of the invention in ethanol (6-8 ml.),add 0.1-0.2% of a lubricating agent, such as polysorbate 85 or oleicacid; and disperse such in a propellant, such as freon, preferably incombination of (1.2 dichlorotetra-fluoroethane) anddifluorochloromethane, and put into an appropriate aerosol containeradapted for either intranasal or oral inhalation administration.

The antibodies and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, i.e., subcutaneously,intramuscularly or intravenously. The compositions for parenteraladministration will commonly comprise a solution of an antibody orfragment thereof of the invention or a cocktail thereof dissolved in anacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be employed, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like. These solutions are sterile and generally free ofparticulate matter. These solutions may be sterilized by conventional,well-known sterilization techniques. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, etc. The concentration of the antibody or fragment thereof ofthe invention in such pharmaceutical formulation can vary widely, i.e.,from less than about 0.5%, usually at or at least about 1% to as much as15 or 20% by weight, and will be selected primarily based on fluidvolumes, viscosities, etc., according to the particular mode ofadministration selected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 Ml sterile buffered water, and50 mg. of an antibody or fragment thereof of the invention. Similarly, apharmaceutical composition of the invention for intravenous infusioncould be made up to contain 250 ml. of sterile Ringer's solution, and150 mg. of an antibody or fragment thereof of the invention. Actualmethods for preparing parenterally administrable compositions are wellknown or will be apparent to those skilled in the art, and are describedin more detail in, for example, Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa., hereby incorporated byreference herein.

The antibodies (or fragments thereof) of the invention can belyophilized for storage and reconstituted in a suitable carrier prior touse. This technique has been shown to be effective with conventionalimmune globulins and art-known lyophilization and reconstitutiontechniques can be employed.

Depending on the intended result, the pharmaceutical composition of theinvention can be administered for prophylactic and/or therapeutictreatments. In therapeutic application, compositions are administered toa patient already suffering from a disease, in an amount sufficient tocure or at least partially arrest the disease and its complications. Inprophylactic applications, compositions containing the presentantibodies or a cocktail thereof are administered to a patient notalready in a disease state to enhance the patient's resistance.

Single or multiple administrations of the pharmaceutical compositionscan be carried out with dose levels and pattern being selected by thetreating physician. In any event, the pharmaceutical composition of theinvention should provide a quantity of the altered antibodies (orfragments thereof of the invention sufficient to effectively treat thepatient.

It should also be noted that the antibodies of this invention may beused for the design and synthesis of either peptide or non-peptidecompounds (mimetics) which would be useful in the same therapy as theantibody. See, e.g., Saragovi et al., Science, 253, 792-795 (1991).

To further illustrate the invention, the following examples areprovided. These examples are not intended, nor are they to be construed,as further limiting the invention.

EXAMPLE 1

Recombinant immunoglobulin libraries displayed on the surface offilamentous phage were first described by McCafferty et al, Nature,348:552-554, 1990 and Barbas et al, Proc. Natl. Acad. Sci., USA88:7978-7982, 1991. Using this technology, high affinity antibodies havebeen isolated from immune human recombinant libraries (Barbas et al,Proc. Natl. Acad. Sci., USA 589:10164-10168, 1992). Although the phagedisplay concept used is substantially similar to that described byBarbas, 1991, Id. the technique has been modified by the substitution ofa unique vector for monkey libraries to reduce the possibility ofrecombination and improve stability. This vector, pMS, FIG. 1 contains asingle lac promoter/operator for efficient transcription and translationof polycistronic heavy and light chain monkey DNA. This vector containstwo different leader sequences, the omp A (Movva et al, J. Biol. Chem.255: 27-29 (1980), for the light chain and the pel B (Lei, J. Bact.,4379-109:4383 (1987) for the heavy chain Fd. Both leader sequences aretranslated into hydrophobic signal peptides that direct the secretion ofthe heavy and light chain cloned products into the periplasmic space. Inthe oxidative environment of the periplasm, the two chains fold anddisulfide bonds form to create stable Fab fragments. We derived thebackbone of the vector from the phagemid bluescript. (Stratagene, LaJolla, Calif.). It contains the gene for the enzyme beta-lactamase thatconfers ampicillin (carbenicillin) resistance to bacteria that harborpMS DNA. We also derived, from bluescript, the origin of replication ofthe multicopy plasmid ColEl and the origin of replication of thefilamentous bacteriophage fl. The origin of replication of phage fl (theso-called intragenic region), signals the initiation of synthesis ofsingle stranded pMS DNA, the initiation of capsid formation and thetermination of RNA synthesis by viral enzymes. The replication andassembly of pMS DNA strands into phage particles requires viral proteinsthat must be provided by a helper phage. We have used helper phageVCSM13 which is particularly suited for this, since it also contains agene coding for kanamycin resistance. Bacteria infected with VCSM13 andpMS can be selected by adding both kanamycin and carbenicillin to thegrowth medium. The bacteria will ultimately produce filamentous phageparticles containing either pMS or VCSM13 genomes. Packaging of thehelper phage is less efficient than that of pMS, resulting in a mixedphage population that contains predominately recombinant pMS phages. Theends of the phage pick up minor coat proteins specific to each end. Ofparticular interest here is the gene III product which is present inthree to five copies at one end of the phage. The gene III product is406 amino acid residues and is required for phage infection of E. colivia the F pili. The first two domains of the heavy chain, the variableand the CH1 domain, are fused to the carboxy-terminal half of the geneIII protein. This recombinant pili protein, directed by the pel Bleader, is secreted to the peroplasm where it accumulates and formsdisulfide bonds with the light chain before it is incorporated in thecoat of the phage. Also, another vector contains a FLAG sequenceengineered downstream of the gene III. The FLAG is an 8 amino acidpeptide expressed at the carboxy terminal of the Fd protein. We areusing commercially available monoclonal anti-FLAG M2 for bothpurification and detection of phage Fab by ELISA (Brizzard, BioTechniques 16(4):730-731 (1994)).

After constructing the vector pMS, we tested its ability to producephage bound Fab using control antibody genes. We cloned an anti-tetanustoxoid antibody, (obtained from Dr. Carlos Barbas), into pMS andtransformed XLI-blue. We co-infected our cells with VCSM13 and generatedphage displaying the anti-tetanus toxoid antibody. We performedefficiency experiments where anti-tetanus toxoid phage were combinedwith phage beading an irrelevant antibody at 1:100,000. We performedthree rounds of panning by applying 50 μl of the mixed phage to antigen(tetanus toxoid) coated polystyrene wells. Non-adherent phage werewashed off and the adherent phage were eluted with acid. The elutedphage were used to infect a fresh aliquot of XL1Blue bacteria and helperphage was added. After overnight amplification, phage were prepared andagain panned on antigen coated plates. After three rounds of panning, wewere able to show that we had successfully enriched for the anti-tetanustoxoid phage. The success of this technology also depends on the abilityto prepare soluble Fabs for characterization of the final pannedproduct. This was achieved by excising gene III from the pMS DNA usingthe restriction enzyme Nhe I followed by re-ligation. After the gene IIIwas excised, the Fab was no longer displayed on the phage surface butaccumulated in the piroplasmic space. Lysates were prepared frombacteria expressing soluble Fab and tested for antigen specificity usingan ELISA. High levels of soluble Fab were detected.

In order to adapt phage display technology for use with macaquelibraries, we developed specific primers for PCR amplifying monkeyimmunoglobulin genes. These were based on macaque sequences we obtainedwhile developing the PRIMATIZED®. antibody technology (See, U.S. Pat.No. 5,658,570) and databases containing human sequences. (Kabat et al,(1991), “Sequences of Proteins of Immunological Interest,” U.S. Dept. ofHealth and Human Services, National Institute of Health).

We developed three sets of primers to cover amplification of the macaquerepertoire. Our first set of primers was designed for amplification ofthe heavy chain VH and CH1 (Fd) domains. It consisted of a 3′ CH1 domainprimer and six 5′ VH family specific primers that bind in the framework1 region. Our second set of primers, for amplifying the whole lambdachain, covers the many lambda chain subgroups. It consists of a 3′primer and three 5′ degenerate primers that bind in the VL framework 1region. Our third set of primers was designed for amplification of thekappa chain subgroups. It consists of one 3′ primer and five VKframework 1 primers. Using each of these sets, PCR parameters wereoptimized to obtain strong enough signals from each primer pair so thatample material was available for cloning of the library. We recentlycreated macaque combinatorial libraries in our pMS vector using theseoptimized PCR conditions. Bone marrow biopsies were taken from CD4immune monkeys as the source of immunoglobulin RNA. The librariescontained approximately 10⁶ members and are currently being panned orspecific binders on antigen coated wells.

EXAMPLE 2 Development of B7/CTLA-4 Reagents

We have generated a number of reagents for the purpose of immunizingmonkeys, developing binding and functional assays in vitro, screeningheterohybridomas and panning phage libraries. Table 1 lists each reagentand its intended purpose. In the case of B7.1, RNA was extracted from SBcells and converted to cDNA using reverse transcriptase. The firststrand cDNA was PCR amplified using B7.1 specific primers and clonedinto IDEC's NEOSPLA mammalian expression vectors, CHO cells weretransfected with B7.1 NEOSPLA DNA and clones expressing membraneassociated B7.1 were identified. The B7.1 fusion protein was generatedsimilarly, except that the PCR amplified B7.1 gene was cloned into aNEOSPLA cassette vector containing the human CH2 and CH3 immunoglobulingenes. CHO cells were transformed with the B7.1 μg NEOSPLA DNA andstable clones secreting B7.1/Ig fusion protein were amplified. Ingeneral, the B7.2 and CTLA4 reagents were generated in the same manner,except that for B7.2 the RNA was isolated from human spleen cells thathad been stimulated 24 hours with anti-Ig and IL-4, and for the CTLA4constructs the gene source was PHA activated human T cells.

TABLE 1 CHO Reagent Purpose Expression Soluble B7.1 Immunization,immunoassays Yes B7.1 Transfectant Screening, ELISA Yes B7.1/Ig FusionProtein Inhibition studies, panning Yes B7.2 Transfectant Screening,ELISA Yes B7.2/Ig Fusion Protein Inhibition studies, panning To becompleted CTLA4 Transfectant Inhibition studies To be completed CTLA4/IgInhibition studies To be completed

The availability of these reagents, together with monoclonal antibodiesto B7.1 (L3074) (Becton Dickinson, 1994) and B7.2 (Fun-1 (Engel et al,Blood, 84, 1402-1407, (1994) and purified goat and rabbit antisera,specifically developed to detect monkey Fab fragments, facilitatesidentification of antibodies having the desired properties.

EXAMPLE 3 Generation of a Phage Display Library

Recombinant phage display libraries are generated from B7.1 and B7.2immune monkeys. Lymph node and bone marrow biopsies are performed 7-12days after immunization to harvest RNA rich B cells and plasma cells.RNA is isolated from the lymphocytes using the method described byChomczynski, Anal. Biochem., 162(1), 156-159 (1987). RNA is converted tocDNA using an oligo dT primer and reverse transcriptase. The firststrand cDNA is divided into aliquots and PCR amplified using the sets ofkappa, lambda, and heavy chain Fd region primers described earlier andeither Pfu polymerase (Stratagene, San Diego) or Taq polymerase(Promega, Madison). The heavy chain PCR amplified products are pooled,cut with Xho VSpe I restriction enzymes and cloned into the vector pMS.Subsequently, the light chain PCR products are pooled, cut with SacI/Xba I restriction enzymes, and cloned to create the recombinantlibrary. XLI-Blue E. coli is transformed with the library DNA andsuper-infected with VCSM13 to produce the phage displaying antibodies.The library is panned four rounds on polystyrene wells coated with B7.1or B7.2 antigen. Individual phage clones from each round of panning areanalyzed. The pMS vector DNA is isolated and the gene III excised.Soluble Fab fragments are generated and tested in ELISA for binding toB7.1 and B7.2.

EXAMPLE 4 Characterization of Phage Fab Fragments

The monkey phage Fab fragments are characterized for their specificityand the ability to block B7.1-Ig and B7.2-Ig binding to CTLA-4-Ig orCTLA-4 transfected cells. Phage fragments are also characterized forcross-reactivity after first panning for 4 rounds on the B7 species usedfor immunization in order to select for high affinity fragments. Fabfragments identified from four rounds of panning either on B7.1 or B7.2antigen coated surfaces are scaled up by infection and grown in 24 hourfermentation cultures of E coli. Fragments are purified by Kodak FLAGbinding to a anti-FLAG affinity column. Purified phage Fabs are testedfor affinity by an ELISA based direct binding modified Scatchardanalysis (Katoh et al, J. Chem. BioEng. 76:451-454 (1993)) using Goatanti-monkey Fab antibodies or anti-FLAG MAb conjugated with horseradishperoxidase. The anti-monkey Fab reagents will be absorbed against humanheavy chain constant region Ig to remove any cross-reactivity to B7-Ig.Kd values are calculated for each fragment after measurements of directbinding to B7.1-Ig or B7.2-Ig coated plates.

EXAMPLE 5 Phage Fab Fragment Blocking of CTLA-4/B7 Binding

Fab fragments most effectively blocking the binding of B7-Ig at thelowest concentrations are selected as lead candidates. Selections aremade by competing off ¹²⁵I-B7-Ig binding to CTLA-4-Ig or CTLA-4transfected cells. Additional selection criteria include, blocking ofmixed lymphocyte reaction (MLR), as measured by inhibiting 3H-thymidineuptake in responder cells (Azuma et al, J. Exp. Med. 177:845-850; Azumaet al., Nature 301:76-79 (1993)) and direct analysis of IL-2 productionusing IL-2 assay kits. The three or four candidates which are mosteffective in inhibiting of MLR and CTLA-4 binding assays are chosen forcloning into the above-described mammalian expression vector fortransfection into CHO cells and expression of chimeric monkey/humanantibodies.

EXAMPLE 6 Generation of Monkey Heterohybridomas

Monkey heterohybridomas secreting monoclonal antibodies are generatedfrom existing immunized animals whose sera tested positive for B7.1and/or B7.2. Lymph node biopsies are taken from animals positive toeither, or both, antigens. The method of hybridoma production is similarto the established method used for the generation of monkey anti-CD4antibodies (Newman, 1992 (Id.)). Monkeys with high serum titers willhave sections of inguinal lymph nodes removed under anesthesia.Lymphocytes are washed from the tissue and fused with KH6/B5heteromyeloma cells (Carrol et al, J. Immunol. Meth. 89:61-72 (1986))using polyethylene glycol (PEG). Hybridomas are selected on H.A.T. mediaand stabilized by repeated subcloning in 96 well plates.

Monkey monoclonal antibodies specific for B7.1 antigen are screened forcross-reactivity to B7.2. Monkey anti-B7 antibodies will becharacterized for blocking of B7/CTLA-4 binding using the ¹²⁵I-B7-Igbinding assay. Inhibition of MLR by 3H-Thymidine uptake and directmeasurement of IL-2 production is used to select three candidates. Twocandidates will be brought forward in Phase II studies and expressed inCHO cells while repeating all functional studies. For the purposes ofdeveloping an animal model for in vivo pharmacology, anti-B7 antibodieswill be tested on cells of several animal species. The establishment ofan animal model will allow preclinical studies to be carried out for theselected clinical indication.

EXAMPLE 7

As discussed supra, using the above heterohybridoma methods, 4 leadmonkey anti-B7.1 antibodies have been identified: 16C10, 7B6, 7C10 and20C9. These antibodies were characterized as follows:

-   -   Scatchard analysis showed that the apparent affinity constants        (Kd) for the monkey antibodies binding to B7-Ig coated plates        were approximated to be:        -   a: 7C10: 6.2×10⁻⁹M        -   b: 16C10: 8.1×10⁻⁹M        -   c: 7B6: 10.7×10⁻⁹M        -   d: 20C9: 16.8×10⁻⁹M    -   The antibodies were tested in vitro in a mixed lymphocyte        reaction assay (MLR). The MLR showed that all 4 anti-B7.1        antibodies inhibit IL-2 production to different extents:        -   a: 7B6: 5.0 μg/Ml        -   b: 16C10: 0.1 μg/Ml        -   c: 20C9: 2.0 μg/Ml        -   d: 7C10: 5.0 μg/Ml    -   The monkey anti-B7.1 antibodies were tested for their ability to        bind B7 on human peripheral blood lymphocytes (PBL). FACS        analysis showed that all 4 monkey antibodies tested positive.    -   Monkey antibodies 16C10, 7B6, 7C10 and 20C9 were tested for C1q        binding by FACS analysis. Results showed 7C10 monkey Ig had        strong human C1q binding after incubating with B7.1        CHO-transfected cells. 16C10 was also positive, while 20C9 and        7B6 monkey antibodies were negative.

EXAMPLE 8

Using the PRIMATIZED® antibody methodology incorporated by reference tocommonly assigned U.S. Pat. No. 5,658,570, and using the NEOSPLA® vectorsystem shown in FIG. 2, the heavy and light chain variable domains of7C10, 7B6 and 16C10 were cloned and PRIMATIZED® forms thereof have beensynthesized in CHO cells using the NEOSPLA® vector system. The aminoacid and nucleic acid sequences for the PRIMATIZED® 7C10 light and heavychain, 7B6 light and heavy chain, and 16C10 light and heavy chain arerespectively shown in FIGS. 3A (SEQ ID NOS: 1-2), 3B-3C (SEQ ID NOS:3-4), 4A (SEQ ID NOS: 5-6), 4B-4C (SEQ ID NOS: 7-8), 5A (SEQ ID NOS:9-10) and 5B-5C (SEQ ID NOS: 11-12).

EXAMPLE 9 Confirming Experiments on the Non-Cross-Reactivity of theCTLA-4 and PRIMATIZED® Antibody Binding Sites on B7.1

In competitive binding assays using biotinylated CTLA-4Ig (FIG. 6),unlabeled PRIMATIZED® 16C10 (i.e., P16C10) was unable to block CTLA-4Igbinding to B7.1 transfected CHO cells. It can be seen that unlabeledCTLA-4Ig and unlabeled B7.1 effectively compete under these conditions.

In a second experiment using Biotinylated P16C10, the same conclusionscan be made. In the experiment shown in FIG. 7, binding of P16C10-Biotinis inhibited by both unlabeled P16C10 and B7.1Ig, but not by CTLA-4Ig.Although CTLA-4Ig is reported to be as much as 4-10 fold higher inaffinity (Kd=0.4 nM; Morton et al., J. Immunol. 156:1047-1054 (1996)),there is no significant inhibition of P16C10 binding even at CTLA-4Igconcentrations as high as 100 fold excess.

Similar results were obtained using the PRIMATIZED® antibody 7C10(P7C10) when it was substituted for P16C10 in the experiments (data notprovided).

EXAMPLE 10 Comparing the Ability of L307.4 and BB-1 Mouse Antibodies toBind to B7 Cho Cells in the Presence of CTLA-4Ig

The binding of L307.4 and BB-1 murine anti-B7 antibody in the presenceof CTLA-4Ig was studied in order to determine whether the mouse antibodybinding sites overlapped with the CTLA-4 binding site. Competition assayexperiments using P16C10-Biotin, L307.4-Biotin and CTLA-4Ig-Biotin weredone to insure that affinity differences did not prevent detection ofcompetitive binding. The results are shown in FIGS. 8 and 9.

The results of FIG. 8 confirm earlier studies that the mouse antibodyBB-1 does not compete with P16C10. These results also show that there issome cross-reactivity to L307.4 of approximately 50%. The results ofFIG. 8 confirm that BB-1 and L307.4 both compete with each other andthat BB-1 completely blocks binding of CTLA-4Ig-Biotin to B7.1transfected CHO cells. BB-1 does not significantly affect P16C10 bindingto B7.1 positive CHO cells.

The results shown in FIG. 9 indicate better than 50% competition whenCTLA-4Ig-Biotin is used in the binding experiment. FIG. 9 shows thatCTLA-4Ig-Biotin is effectively blocked by all B7.1 inhibitors exceptP16C10, therefore P16C10 recognizes a unique binding determinate on B7.1which allows the normal CTLA-4 ligand binding in the generation ofnegative signals. Earlier functional studies (data not shown) suggest aweakened ability of L307.4 to block IL-2 production in allogeneic MLR,which correlates with the hypothesis that it may interfere with CTLA-4negative signaling. It is not clear how many of the other murineantibodies reported in the literature give complete inhibition of CTLA-4binding; however, this issue may be important in defining the truefunctional mechanisms of B7.1 and B7.2 specific antibodies.

These results confirm earlier studies using B7-Ig in competition withP16C10-Biotin for binding to B7.1 transfected CHO cells. The studiesalso confirm earlier observations of no inhibition of the P16C10 byCTLA-4Ig. These results are highly suggestive that the primateantibodies are specific for a unique B7.1 epitope independent of theCTLA-4 binding site which interacts primarily with CD28. This type ofinteraction would provide a benefit, since it has the ability to blockbinding of B7.1 to CD28 receptors while still allowing the negativesignaling function of CTLA-4 to occur uninhibited. This perceivedinteraction may lead to a down regulation of the overall T cellactivation response regardless of the predominance of either Th1 or Th2phenotypes.

Similar results were obtained using P7C10 when it was substituted forP16C10 in the experiments (data not provided).

EXAMPLE 11 Experiment Demonstrating the Ability of P16C10 to Bind andBlock B7.1 Interactions with CD28 Receptor

An experiment to determine if P16C10 binding of B7.1 can block theinteraction of B7.1 with CD28 was attempted by radiolabeling B7.11 gwith ¹²⁵I, followed by competitive binding to CD28 positivenon-activated peripheral blood T lymphocytes. The results shown in FIG.10 demonstrate that the radiolabeled B7.11 g binds specifically to the Tcells, as confirmed by inhibition with unlabeled B7. Ig. The resultsalso show that CTLA-4Ig, anti-CD28 and P16C10 are all capable ofblocking this interaction. The results further confirm that P16C10blocks binding of the CD28/B7 interaction with an IC₅₀ of <1 ug/mL.

The above results were obtained under conditions where no membraneassociated CTLA-4 was expressed (Linsley et al., J. Exp. Med.173:721-730 (1991)) and confirmed by blocking with the anti-CD28antibody.

Similar results were obtained using P7C10 when it was substituted forP16C10 in the experiments (data not provided).

EXAMPLE 12 IDEC-114 does not Block IL-2 Production in Co-Stimulated TCells

In an experiment, the results of which are contained in FIG. 11, asub-optimal primary signal was induced by attaching an anti-CD3 antibodyand a soluble B7Ig fusion protein to covalently coupled protein-A latexmicrospheres. Initially, a 1:10 ratio of anti-CD3 to B7Ig was used whichis a relatively high density of B7 co-stimulatory molecules that isseveral times greater than normal cells express based on the relativeamounts of IL-2 that are typically produced. Purified CD4+ T cellsobtained from blood bank donors and co-cultured the cells in presence orabsence of soluble CD28:B7 inhibitors that included anti CD80 antibodiesL307.4 and IDEC-114 and soluble CTLA-4Ig fusion protein were added atthree concentrations ranging from 10 to 0.1 μg/mL. Samples of tissueculture media were collected after 48 hours and the IL-2 cytokinepresent in the cultures was determined. The results show clearly thatbeads containing anti-CD3 alone and B7Ig alone produced little or noIL-2. By contrast, both anti-CD3 and B7Ig were present approximately4500 pg/mL of IL-2 was produced. The results also revealed that bothL307.4 and CTLA-4Ig completely inhibited the production of IL-2 at allconcentrations where IDEC-114 had no effect. These results suggest thatL307.4 and CTLA-4Ig regulate the activation of T cells by a similarmechanism that directly interferes with CD28 signaling to produce theactivating cytokine IL-2 while IDEC-114 has no such functional property.

EXAMPLE 13 IDEC-114 does not Block Growth in Co-Stimulated T Cells

The same cultures were analyzed for effect on cell growth and similarresults to the effects on IL-2 production were obtained. As seen in FIG.12, L307.4 and CTLA-4Ig were equally effective in totally blocking cellproliferation as determined by uptake of radiolabeled thymidine. Underthese same conditions, IDEC-114 had no effect on cell growth. Theseresults further suggest that IDEC-114 is not directly regulating thegrowth and differentiation properties of CD4+ T cells by blocking theinteraction between B7(CD80) and CD28 receptors, unlike other testedanti-B7 antibodies having different binding specifications.

EXAMPLE 14 IDEC-114 Partially Blocks IL-10 Production in Co-Stimulated TCells

The same cultures were analyzed for the presence of the secondary TH2cytokine IL-10. It was found that both L307.4 and CTLA-4 againcompletely blocked IL-10 production, whereas IDEC-114 only partiallyblocked IL-10 proeduction. As seen in FIG. 3, L307.4 and CTLA-4Ig wereabout equally effective in blocking IL-10 production while IDEC-114 hada partial effect. The partial inhibition of IL-10 by IDEC-114 may be afunction of its property of allowing negative signaling to occur in Tcells by not interfering with the function of CTLA-4 expressed in Tcells. CTLA-4 is upregulated in T cells during co-stimulation and isthought to provide a negative signal to T cells. These results furthersuggest that IDEC-114 is not regulating the cytokine producingproperties of CD4+ T cells through the normal channels involving CD28signal transduction.

EXAMPLE 15 IDEC-114 Blocks IL-2 Production in T Cells Co-Stimulated withMicro-Beads Containing Reduced Amounts of B7

In another experiment, the ratio of anti-CD3 to B7-Ig was adjusted onthe stimulator beads from a 1:10 ratio to 8:1 or a reduction in B7 ofabout 80-fold with an 8-fold excess of anti-CD3. The production of IL-2under these conditions is significantly reduced to typically less than1000 pg/mL and is more in line with cultures stimulated with mismatchedallotypes or CD80 transfected cells. Under these conditions (FIG. 14),we observed near complete inhibition of IL-2 with CTLA-4Ig consistentwith results obtained by beads with an anti-CD3/B7-Ig ratio of 1:10.However, with IDEC-114, we routinely observed significant inhibition ofIL-2 (50-90%). We are also able to block IL-2 generated in cultures ofmixed lymphocytes or when B7 transfected CHO cells are used asstimulators instead of microbeads.

These results suggest that IDEC-114 may function by interfering withadhesion and its effects may be facilitated by reduced expression ormaintaining of a lower avidity form of CD80 during co-stimulation.

Analysis of Results

The T cell regulatory properties of IDEC-114 and CTLA-4Ig were comparedin an in vitro co-stimulatory system that includes purified CD4+ helperT cells in the absence of accessory cells. In place of accessoryantigen-presenting cells, Protein A coated latex microspheres andattached anti-CD3 and B7Ig fusion protein were used. When T-cells wereincubated with beads that contained a 10-fold excess of B7 there was astrong co-stimulatory response as measured by IL-2, IL-10 and cellgrowth that was totally blocked by CTLA-4Ig and a commercially availableanti-CD80 monoclonal antibody L307.4. By contrast IDEC-114 had no effecton IL-2 or cell growth but did partially inhibit IL-10 production. Itappears that both CTLA-4 and L307.4 possess higher affinities to B7antigen and that increasing the concentration of IDEC-114 in thecultures should result in the same effect. Based thereon, the affinitiesof CTLA-4Ig and IDEC-114 (Kd=4 nM) were compared by surface plasmonresonance. It was found that the affinity of CTLA-4Ig (Morten et al.,1996, J. Immunol. 156: 1047-1054) was approximately 10-fold higher(Kd=0.4 nM). This assay was performed using as much as 1000-fold greaterconcentration of IDEC-114 with no effect on IL-2. Also, when T-cellswere activated in cultures with beads containing a reduced content of B7(anti-CD3/B7, 8:1), significant blocking of IL-2 production by bothCTLA-4Ig and IDEC-114 was observed. In the latter experiment theequivalent blocking ability of IDEC-114 required approximately 10-foldhigher concentration than CTLA-4Ig and was comparable to the differencebetween the affinities of the two. These results were interpreted byhypothesizing that reduced amounts of B7 on the beads may lead to areduction in the forming of stable interactions with CD28. This lowaffinity state may somewhat resemble normal resting B cells. Therefore,with the reduced avidity for adhesion, IDEC-114 may bind to a remotesite inducing a conformational change resulting in even lower affinityof CD80 for CD28.

Alternatively, antigen presenting cells that become activated mayincrease their surface density to the extent that the highly mobile B7molecules more easily form homodimers. With respect thereto, it has beenreported that monomeric forms of B7 have extremely low affinity and fastoff-rate kinetics (van der Merwe, et al. 1997, J. Exp. Med., 185:393-403) and that homodimeric forms can have up to 500-fold higheraffinity. A higher affinity form would understandably facilitate clusteror patch formation leading to a more stable receptor ligand complex.Consequently, IDEC-114 may bind to a remote site that could restrict theassociation of neighboring CD80 molecules and reduce or limit the amountof dimerization effectively limiting the adhesion complex formation.

The observed results suggest that PRIMATIZED® antibodies having thenovel binding properties disclosed herein do not influence theregulation of T cells, unlike prior anti-B7.1 antibodies. It ishypothesized, based on these results, that the antibodies of theinvention, such as IDEC-114, bind to a unique site on CD80 expressed onantigen presenting cells which prevents the association of CD80receptors from forming a higher affinity interaction with CD28 receptorson T cells. This would generate a weaker signal through CD28 that undercertain conditions could not be overcome by the upregulation of more B7ligand. However, these same results do not preclude the ability ofIDEC-114 to function in other types of immune regulatory mechanismsoccurring in vivo where the presence of NK cells and macrophages maycontribute to killing of B cells or activated T cells through Fe andcomplement mediated effects.

It is anticipated that these PRIMATIZED® antibodies, given theirprobable low antigenicity and human effector function, will be wellsuited as therapeutics. In this regard, it has been shown thatPRIMATIZED® 16C10 (IDEC-114) exhibits human C1q binding.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be embraced by the following claims.

DEPOSIT INFORMATION

Hybridoma 7B6, hybridoma 7C10, and hybridoma 16C10, which produceantibodies 7C10 and 16C10, respectively, were deposited on May 29, 1996,with the American Type Culture Collection (ATCC), currently located at10801 University Boulevard, Manassas, Va. 20110-2209, under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganisms for the Purpose of Patent Procedure(“Budapest Treaty”). The ATCC has assigned hybridoma 7B6 the ATCCAccession No. HB-12120, assigned hybridoma 7C10 the ATCC Accession No.HB-12117, and assigned hybridoma 16C10 the ATCC Accession No. HB-12119.

1. A method of treating rheumatoid arthritis in a subject in need ofsuch treatment comprising administering a therapeutically effectiveamount of a monoclonal anti-CD80 antibody or CD80 binding fragmentthereof that binds specifically to human CD80 antigen and inhibits thebinding of the CD80 antigen to CD28 without inhibiting the binding ofthe CD80 antigen to CTLA-4.
 2. The method of claim 1, wherein theantibody competes for binding to human CD80 antigen with a 7C10antibody, produced by a hybridoma assigned ATCC Accession No. HB-12117,or a 16C10 antibody, produced by a hybridoma assigned ATCC Accession No.HB-12119.
 3. The method of claim 1, wherein the antibody is a humanmonoclonal antibody or a chimeric or humanized antibody comprising humanconstant regions.
 4. The method of claim 3, wherein the antibody is achimeric antibody comprising variable regions of an Old World monkeyantibody and human constant regions.
 5. The method of claim 4, whereinthe antibody comprises variable regions of a 7C10 antibody, produced bya hybridoma assigned ATCC Accession No. HB-12117, or variable regions ofa 16C10 antibody, produced by a hybridoma assigned ATCC Accession No.HB-12119.
 6. The method of claim 4, wherein the antibody comprises ahuman constant region selected from the group consisting of a humangamma 1 constant region, a human gamma 4 constant region, and a humangamma 4PE constant region.
 7. The method of claim 4, wherein theantibody comprises a light chain variable region having an amino acidsequence contained in SEQ ID NO: 2 and a heavy chain variable regionhaving an amino acid sequence contained in SEQ ID NO:
 4. 8. The methodof claim 4, wherein the antibody comprises a light chain variable regionhaving an amino acid sequence contained in SEQ ID NO: 10 and a heavychain variable region having an amino acid sequence contained in SEQ IDNO:
 12. 9. The method of claim 5, wherein the antibody comprisesvariable regions of a 16C10 antibody, produced by a hybridoma assignedATCC Accession No. HB-12119.
 10. The method of claim 9, wherein theantibody comprises a human gamma 1 constant region.
 11. The method ofclaim 1, wherein the antibody fragment is selected from the groupconsisting of Fab, F(ab′)₂ and Fv.
 12. The method of claim 1, whereinthe antibody has the same epitopic specificity as an antibody selectedfrom the group consisting of: (a) an antibody having as its light chainvariable region and heavy chain variable region sequences the variableregion sequences contained in SEQ ID NO: 2 and SEQ ID NO: 4,respectively; and (b) an antibody having as its light chain variableregion and heavy chain variable region sequences the variable regionsequences contained in SEQ ID NO: 10 and SEQ ID NO: 12, respectively.13. A method of treating rheumatoid arthritis in a subject in need ofsuch treatment comprising administering a therapeutically effectiveamount of a monoclonal anti-CD80 antibody or CD80 binding fragmentthereof, wherein the antibody specifically binds to human CD80 antigenand comprises the light chain variable region and heavy chain variableregion sequences contained in SEQ ID NO: 10 and SEQ ID NO: 12,respectively, and human light chain and heavy chain constant regions.14. The method of claim 13, wherein the antibody comprises a human gamma1 or gamma 4 constant region.
 15. The method of claim 13, wherein theantibody comprises light chain and heavy chain sequences set forth asSEQ ID NO: 10 and SEQ ID NO:12, respectively.
 16. A method of treatingrheumatoid arthritis in a subject in need of such treatment comprisingadministering a therapeutically effective amount of a monoclonalanti-CD80 antibody or CD80 binding fragment thereof, wherein theantibody specifically binds to human CD80 antigen and comprises a lightand heavy chain variable domain from a 16C10 antibody produced by ahybridoma assigned ATCC Accession No. HB-12119 and human constantregions.
 17. The method of claim 16, wherein the human constant regionis a human gamma 1 or gamma 4 constant region.
 18. The method of claim16, wherein the antibody is produced by a hybridoma assigned ATCCAccession No. HB-12119.
 19. The method of claim 1, further comprisingadministering an immunomodulator selected from the group consisting ofIL-7, IL-10, CTLA4-Ig, soluble CTLA-4, an anti-CD28 antibody, and aCD28-binding fragment of an anti-CD28 antibody.
 20. The method of claim1, further comprising administering an immunosuppressant selected fromthe group consisting of cyclosporine A, FK506, anti-TNFα antibody,anti-CD54 antibody, anti-CD11 antibody, anti-CD11a antibody, anti-IL-1antibody, TNFα receptor, and IL-1 receptor, wherein the anti-CD80antibody or CD80 binding fragment thereof.